Multipotent stem cells from peripheral tissues and uses thereof

ABSTRACT

This invention relates to multipotent stem cells, purified from the peripheral tissue of mammals, and capable of differentiating into neural and non-neural cell types. These stem cells provide an accessible source for autologous transplantation into CNS, PNS, and other damaged tissues.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/099,539(now issued U.S. Pat. No. 7,544,509), filed Mar. 15, 2002, which is acontinuation-in-part of and claims priority to U.S. application Ser. No.09/991,480, filed Nov. 9, 2001 (abandoned), which application is acontinuation-in-part of U.S. application Ser. No. 09/916,639, filed Jul.26, 2001 (abandoned), which application is a continuation-in-part of PCTapplication CA01/00047 filed Jan. 24, 2001 and to U.S. application Ser.No. 09/670,049 (now issued U.S. Pat. No. 6,787,355), filed Sep. 25,2000, which application is a continuation-in-part of application Ser.No. 09/490,422, filed Jan. 24, 2000 (abandoned), each of which is herebyincorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to multipotent stem cells (MSCs) purifiedfrom peripheral tissues including peripheral tissues containing sensoryreceptors such as skin, olfactory epithelium, mucosa, and tongue. Theinvention also relates to cells differentiated from these multipotentstem cells. The invention includes pharmaceutical compositions and usesof either the multipotent stem cells or the differentiated cells derivedfrom such stem cells. Additionally, business methods based on themultipotent stem cells or the differentiated cells are contemplated.

There are a number of diseases of the central nervous system (“CNS”)which have a devastating effect on patients. These diseases aredebilitating, often incurable, and include, for example, Alzheimer'sdisease, Huntington's disease, Parkinson's disease, and MultipleSclerosis.

By way of example, Parkinson's disease is a progressive degenerativedisorder of unknown cause. In healthy brain tissue, dopaminergic neuronsextend from the substantia nigra of the brain into the neighboringstriatum. In Parkinson's disease, these dopaminergic neurons die.

There are a number of methods to treat Parkinson's disease. One methodis to treat humans having Parkinson's disease with L-DOPA. A secondmethod is to transplant cells into the substantia nigra or striatum.Transplanted cells replace endogenous cells that are lost as aconsequence of disease progression. An animal model of Parkinson'sdisease is an MPTP-treated non-human primate. The MPTP-treated animalshave been transplanted with dopamine-rich embryonic neurons with somesuccess.

To date, the cells used for neural transplant have been collected fromthe developing brains of aborted fetuses. Aside from the ethicalconsiderations, the method from a practical standpoint is unlikely toprovide a sufficient amount of neural tissue to meet the demands. Thus,another source of cells for transplantation is desirable.

Stem cells are undifferentiated cells that exist in many tissues ofembryos and adult organisms. In embryos, blastocyst stem cells are thesource of cells which differentiate to form the specialized tissues andorgans of the developing fetus. In adults, specialized stem cells inindividual tissues are the source of new cells, replacing cells lostthrough cell death due to natural attrition, disease, or injury. Stemcells may be used as substrates for producing healthy tissue where adisease, disorder, or abnormal physical state has destroyed or damagednormal tissue.

Weiss et al., 1996 summarizes the five defining characteristics of stemcells as the ability to:

-   -   Proliferate: Stem cells are capable of dividing to produce        daughter cells.    -   Exhibit self-maintenance or renewal over the lifetime of the        organism: Stem cells are capable of reproducing by dividing        symmetrically or asymmetrically to produce new stem cells.        Symmetric division occurs when one stem cell divides into two        daughter stem cells. Asymmetric division occurs when one stem        cell forms one new stem cell and one progenitor cell. Symmetric        division is a source of renewal of stem cells. This permits stem        cells to maintain a consistent level of stem cells in an embryo        or adult mammal.    -   Generate large number of progeny: Stem cells may produce a large        number of progeny through the transient amplification of a        population of progenitor cells.    -   Retain their multilineage potential over time: Stem cells are        the ultimate source of differentiated tissue cells, so they        retain their ability to produce multiple types of progenitor        cells, which will in turn develop into specialized tissue cells.    -   Generate new cells in response to injury or disease: This is        essential in tissues which have a high turnover rate or which        are more likely to be subject to injury or disease, such as the        epithelium of blood cells.

Thus, the key features of stem cells are that they are multipotentialcells which are capable of long-term self-renewal over the lifetime of amammal.

MSCs may be used as a source of cells for transplantation. The stemcells may themselves be transplanted or, alternatively, they may beinduced to produce differentiated cells (e.g., neurons,oligodendrocytes, Schwann cells, or astrocytes) for transplantation.Transplanted stem cells may also be used to express therapeuticmolecules, such as growth factors, cytokines, anti-apoptotic proteins,and the like. Thus, stem cells are a potential source of cells foralternative treatments of diseases involving loss of cells or tissues.

The safest type of tissue graft (using stem cells or otherwise) is onethat comes from self (an autologous tissue source). Autologous tissuesources are widely used in procedures such as bone transplants and skintransplants because a source of healthy tissue is readily accessible fortransplant to a damaged tissue site. In brain diseases, such asParkinson's disease, healthy dopaminergic neuronal brain tissue mayexist at other sites in the brain, but attempts to transplant theseneurons may harm the site where the healthy neurons originate.Multipotent stem cells that can be differentiated into dopaminergicneurons may be available at other sites from which they may betransplanted, but the CNS, particularly the brain, is physicallydifficult to access.

In several tissues, stem cells have been purified and characterized. Forexample, neural stem cells have been purified from the mammalianforebrain (Reynolds and Weiss, Science 255:1707-1710, 1992) and thesecells were shown to be capable of differentiating into neurons,astrocytes, and oligodendrocytes. PCT publications WO 93/01275, WO94/16718, WO 94/10292 and WO 94/09119 describe uses for these cells. Itcould be impractical or impossible, however, to first access brain orother CNS tissue for biopsy and then again for transplant in patientswith weakened health. It would be very useful if there were accessiblestem cells capable of differentiating into CNS cell types, such asdopaminergic neurons; such cells would be a source of cells forautologous transplants.

Thus, there is a clear need to develop methods for identifying fromaccessible tissues multipotent stem cells that can act as a source ofcells that are transplantable to the CNS, PNS, or other tissues in vivoin order to replace damaged or diseased tissue.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to preparations of purifiedmultipotent stem cells that are obtained from peripheral tissue ofmammals, preferably from postnatal mammals such as juvenile and adultmammals. We have identified epithelial tissues, such as skin, asconvenient sources of multipotent stem cells, and provide methods forthe purification of epithelial-derived MSCs, thus simplifying theharvesting of cells for transplantation relative to previous methods.The MSCs possess desirable features in that they are multipotent andself-renewing. The cells can be repeatedly passaged and can bedifferentiated into numerous cell types of the body includingderivatives of ectodermal and mesodermal tissue. The MSCs of thisinvention are positive for nestin protein, an immunological marker ofstem cells and progenitor cells, as well as fibronectin protein, but arenegative for vimentin and cytokeratin when assayed byimmunohistochemistry. Moreover, the MSCs of the present invention growas non-adherent clusters when cultured by the methods herein disclosed,and one of skill in the art will readily recognize that such cells willgrow as non-adherent clusters when cultured on a variety of substratumincluding but not limited to uncoated plastic or plastic coated with aneutral substrate such as gelatin or agar. In certain embodiments, theMSCs of this invention are negative for the neural crest stem cellmarker p75. These characteristics distinguish the cells of thisinvention from other stem cells, including mesenchymal stem cells andneural crest stem cells.

In certain embodiments, the cells are capable of differentiating asdopaminergic neurons, and thus are a useful source of dopaminergicneurons for homotypic grafts into Parkinson's Disease patients. The MSCscan also differentiate as numerous mesodermal derivatives includingsmooth muscle cells, adipocytes, cartilage, bone, skeletal muscle, andcardiac muscle, and are expected to be capable of producing othermesodermal derivatives including kidney and hematopoietic cells.Additionally, we show that the MSCs can express markers of endodermaldifferentiation, and are expected to differentiate to cell typesincluding pancreatic islet cells (e.g., alpha, beta, phi, delta cells),hepatocytes, and the like. To our knowledge, the MSCs of the inventionrepresent the first adult stem cell capable of differentiating to cellderived from all three germ layers. The subject cells may also be usedfor autologous or heterologous transplants to treat, for example, otherneurodegenerative diseases, disorders, or abnormal physical states.

Accordingly, in a first aspect, the invention features MSCssubstantially purified from a peripheral tissue of a postnatal mammal.In preferred embodiments, the peripheral tissue is an epithelial tissueincluding skin or mucosal tissue. In a second embodiment, the peripheraltissue is derived from the tongue. In still another embodiment, thetissue is derived from skin. The postnatal mammal may be either ajuvenile or adult mammal.

In certain embodiments, the invention features a cell that is theprogeny of a MSC substantially purified from a peripheral tissue of apostnatal mammal. The cell may be a mitotic cell or a differentiatedcell (e.g., a neuron, an astrocyte, an oligodendrocyte, a Schwann cell,or a non-neural cell). Preferred neurons include neurons expressing oneor more of the following neurotransmitters: dopamine, GABA, glycine,acetylcholine, glutamate, and serotonin. Preferred non-neural cellsinclude cardiac muscle cells, pancreatic cells (e.g., islet cells(alpha, beta, phi and delta cells), exocrine cells, endocrine cells,chondrocytes, osteocytes, skeletal muscle cells, smooth muscle cells,hepatocytes, hematopoietic cells, and adipocytes. These non-neural celltypes include both mesodermal and endodermal derivatives. In a preferredembodiment, the differentiated cells are substantially purified.

In a second aspect, the invention features a population of at least tencells, wherein at least 30% of the cells are MSCs substantially purifiedfrom a peripheral tissue of a postnatal mammal or progeny of the MSCs.

Preferably, at least 50% of the cells are MSCs substantially purifiedfrom the peripheral tissue or progeny of the MSCs. More preferably, atleast 75% of the cells are MSCs substantially purified from theperipheral tissue or progeny of the MSCs. Most preferably, at least 90%,95%, or even 100% of the cells are MSCs substantially purified from theperipheral tissue or progeny of the MSCs. The MSCs may be cultured forextended periods of time. Thus, the population of cells may have been inculture for at least thirty days, sixty days, ninety days, or longer(e.g., one year or more). Preferably, the population is at least twentycells, and may be more than fifty cells, a thousand cells, or even amillion cells or more.

In a third aspect, the invention features preparations of at least tencells, and more preferably at least 10⁴, 10⁵, 10⁶ or even 10⁷ cells,having less than 25% lineage committed cells. Preferably, less than 20%of the cells are lineage committed cells. More preferably, less than 15%of the cells are lineage committed cells. Most preferably, less than10%, 5%, or even 0% of the cells are lineage committed cells. Ingeneral, any cell feeder layer upon which the cells of the invention arecultured would not be considered in such a calculation.

In a fourth aspect, the invention features a pharmaceutical compositionincluding (i) a mitotic or differentiated cell that is the progeny of aMSC substantially purified from a peripheral tissue of a postnatalmammal, and (ii) a pharmaceutically acceptable carrier, auxiliary orexcipient.

In a fifth, related aspect, the invention features a pharmaceuticalcomposition including (i) a MSC substantially purified from a peripheraltissue of a postnatal mammal, and (ii) a pharmaceutically acceptablecarrier, auxiliary or excipient.

Preferably, the composition of the fourth or fifth aspect includes apopulation of cells, wherein at least 30%, 50%, 75%, 90%, 95%, or even100% of the cells are MSCs substantially purified from the peripheraltissue or progeny of the MSCs. The composition may include one or moretypes of cells selected from a group consisting of MSCs, or neurons,oligodendrocytes, Schwann cells, astrocytes, adipocytes, smooth musclecells, cardiomyocytes, chondrocytes, osteocytes, skeletal muscle cells,hepatocytes, hematopoietic cells, exocrine cells, endocrine cells andalpha, beta, phi and delta cells, which are progeny of MSCs.

In a sixth aspect, the invention features a method of producing apopulation of at least ten cells, wherein at least 30% of the cells areMSCs substantially purified from a peripheral tissue of a postnatalmammal or progeny of the MSCs: (a) providing the peripheral tissue fromthe mammal; (b) culturing the tissue under conditions in which MSCsproliferate and in which at least 25% of the cells that are not MSCsdie; and (c) continuing culture step (b) until at least 30% of the cellsare MSCs or progeny of the MSCs.

In a seventh aspect, the invention features another method of producinga population of at least ten cells, wherein at least 30% of the cellsare MSCs substantially purified from skin tissue of a postnatal mammalor progeny of the MSCs, the method including: (a) providing the skintissue from the mammal; (b) culturing the tissue under conditions inwhich MSCs proliferate and in which at least 25% of the cells that arenot MSCs die; (c) separating the MSCs from cells that are not MSCs basedon the tendency of MSCs to form non-adherent clusters; and (d) repeatingsteps (b) and (c) until at least 30% of the cells are MSCs or progeny ofthe MSCs.

Suitable culture conditions for step (b) of the sixth and seventhaspects are preferably as follows: (i) triturating or otherwiseseparating tissue into single cells or cell clusters and placing intoculture medium; (ii) culturing the cells in culture medium and underconditions (e.g., DMEM: Ham's F-12 medium containing B-27 supplement,antibacterial and antifungal agents, 5-100 ng/ml bFGF, and 2-100 ng/mlEGF) that allows for the proliferation of MSCs but does not promote, tothe same extent, proliferation of cells that are not MSCs; and (iii)culturing the separated tissue for three to ten days, during which timethe MSCs proliferate in suspension and form non-adherent clusters butnon-MSCs do not proliferate in suspension (these cells either attach tothe plastic or they die). Preferably, at least 50% of the cells insuspension surviving after the period in culture are MSCs or progeny ofthe MSCs, more preferably, at least 75% of the cells are MSCs or progenyof the MSCs, and, most preferably, at least 90% or even 95% of thesurviving cells are MSCs or progeny of the MSCs. In preferredembodiments, tissue is separated mechanically.

In an eighth aspect, the invention features a method of treating apatient having a disease associated with cell loss. In one embodiment,the method includes the step of transplanting the multipotent stem cellsof the invention into the region of the patient in which there is cellloss. Preferably, prior to the transplanting step, the method includesthe steps of providing a culture of peripheral tissue and isolating amultipotent stem cell from the peripheral tissue. The tissue may bederived from the same patient (autologous) or from either a geneticallyrelated or unrelated individual. After transplantation, the method mayfurther include the step of differentiating (or allowing thedifferentiation of) the MSCs into a desired cell type to replace thecells that were lost. Preferably, the region is a region of the CNS orPNS, but can also be cardiac tissue, pancreatic tissue, or any othertissue in which cell transplantation therapy is possible. In a secondembodiment, the method includes the step of delivering the stem cells tothe site of cell damage via the bloodstream, wherein the stem cells hometo the site of cell damage. In a third embodiment, the method fortreating a patient includes the transplantation of the differentiatedcells which are the progeny of the stem cells of this invention.

In a ninth aspect, the invention features a kit including MSCssubstantially purified from peripheral tissue of a postnatal mammal, ora mitotic or differentiated cell that is the progeny of the MSC,preferably wherein the peripheral tissue from which the MSC is purifiedincludes a sensory receptor. Preferably, the kit includes a populationof cells, wherein at least 30%, 50%, 75%, 90%, or even 95% of the cellsare MSCs substantially purified from the peripheral tissue or progeny ofthe MSCs.

In a tenth aspect, the invention features a kit for purifying MSCs fromperipheral tissue. The kit includes media or media components that allowfor the substantial purification of MSCs of the present invention. Thekit may also include media or media components that allow for thedifferentiation of the MSCs into the desired cell type(s). Preferably,the kit also includes instructions for its use. In one embodiment, themedia includes one or more therapeutic proteins, pharmaceutical agents,and/or small molecules that influence the proliferation,differentiation, and/or survival of the MSCs.

In one preferred embodiment of each of the foregoing aspects of theinvention, the peripheral tissue is skin tissue. In another preferredembodiment, the peripheral tissue is tongue tissue, hair follicles,sweat glands, or sebaceous glands. In another preferred embodiment ofeach of the foregoing aspects of the invention, the stem cells arenegative for p75.

The peripheral tissue can be from a newborn mammal, a juvenile mammal,or an adult mammal. Preferred mammals include, for example, humans,non-human primates, mice, pigs, and rats. The MSCs can be derived fromperipheral tissue of any individual, including one suffering from adisease or from an individual immunologically compatible to anindividual suffering from a disease. In a preferred embodiment, thecells, or progeny of the cells, are transplanted into the CNS or PNS ofan individual having a neurodegenerative disease and the individual isthe same individual from whom the MSCs were purified. Followingtransplantation, the cells can differentiate into cells that are lackingor non-functional in the disease.

Preferably, the MSCs are positive for nestin and fibronectin protein andmay also express glutamic acid decarboxylase, but are negative forvimentin and cytokeratin protein. The MSCs of the present invention can,under appropriate conditions, differentiate into neurons, astrocytes,Schwann cells, oligodendrocytes, and/or non-neural cells (e.g., cardiacmuscle cells, skeletal muscle cells, pancreatic cells, smooth musclecells, adipocytes, hepatocytes, cartilage, bone, etc.). In a preferredembodiment, the differentiated neurons are dopaminergic neurons. In apreferred embodiment, the differentiated non-neural cells are selectedfrom smooth muscle cells, adipocytes, cartilage, bone, skeletal muscle,or cardiac muscle.

We show that the MSCs of the invention have tremendous capacity todifferentiate into a range of neural and non-neural cell types. Thenon-neural cell types include both mesodermal and endodermalderivatives. The MSCs of the invention thus provide an adult stem cellcapable of differentiating to derivatives of all three germ layers. Thiscapacity can be further influenced by modulating the culture conditionsto influence the proliferation, differentiation, and survival of theMSCs. In one embodiment, modulating the culture conditions includesincreasing or decreasing the serum concentration. In another embodiment,modulating the culture conditions includes increasing or decreasing theplating density. In still another embodiment, modulating the cultureconditions includes the addition of one or more pharmacological agentsto the culture medium. In another embodiment, modulating the cultureconditions includes the addition of one or more therapeutic proteins(i.e., growth factors, cytokines, anti-apoptotic proteins) to theculture medium. In still another embodiment, modulating the cultureconditions includes the addition of one or more small molecules thatagonize or antagonize the function of a protein involved in cellproliferation, differentiation, or survival. In each of the foregoingembodiments, pharmacological agents, therapeutic proteins, and smallmolecules can be administered individually or in any combination, andcombinations of any of the pharmaceutical agents, therapeutic proteins,and small molecules can be co-administered or administered at differenttimes.

MSCs can be stably or transiently transfected with a heterologous gene(e.g., one encoding a therapeutic protein, such as a protein whichenhances cell divisions or prevents apoptosis of the transformed cell orother cells in the patient, or a cell fate-determining protein). In oneembodiment, the heterologous gene modulates one or more of cellproliferation, differentiation, or survival. In preferred embodiments,transfection of the heterologous gene is adenoviral mediated. In anotherpreferred embodiment, transfection occurs using standard protocols fortransfection in cell culture including lipofectamine mediatedtransfection or electroporation.

In an eleventh aspect, the invention features preparations of stem cellsand their differentiated progeny preserved for subsequent retrieval. Inone preferred embodiment, the preserved cells are formulated in apharmaceutically acceptable carrier. In another embodiment, the stemcells or differentiated progeny are preserved using cryogenic methods.

In a twelfth aspect, the invention features a method for conducting aregenerative medicine business. In one embodiment, the method comprisesaccepting and cataloging tissue samples from a client, culturing thecells from said sample to expand the multipotent stem cells, preservingsuch cells and storing them for later retrieval. In a second embodiment,the method comprises accepting and cataloging tissue samples from aclient, culturing the cells from said sample to expand the multipotentstem cells, and differentiating the stem cell. Both of these embodimentsalso contemplate a billing system for billing the client or an insuranceprovider.

In a thirteenth aspect, the invention features a method for conducting astem cell business comprising identifying agents which influence theproliferation, differentiation, or survival of the multipotent stemcells of the invention. Such agents include small molecules andextracellular proteins. In a preferred embodiment, the identified agentscould be profiled and assessed for safety and efficacy in animals. Inanother preferred embodiment, the invention contemplates methods forinfluencing the proliferation, differentiation, or survival of themultipotent stem cells of the invention by contacting the cells with anagent or agents identified by the foregoing method. In another preferredembodiment, the identified agents are formulated as a pharmaceuticalpreparation. This pharmaceutical preparation can be manufactured,marketed, and distributed for sale.

In a fourteenth aspect, the invention includes a method for conducting adrug discovery business comprising identifying factors which influencethe proliferation, differentiation, or survival of the multipotent stemcells of the invention, and licensing the rights for furtherdevelopment.

In the foregoing aspects of the invention, it is appreciated that theMSCs of the invention can proliferate in culture, and differentiate toderivatives of all three germ layers. Therefor, the MSCs provide novelcompositions of adult stem cells which have therapeutic applications intreating conditions which affect wide range of cell types derived fromall three germ layers. Recognizing the ability of these cells todifferentiate to derivatives of all three germ layers, in a fifteenaspect, the invention includes a cellular composition of adult stemcells which (i) will proliferate in an in vitro culture, (ii) maintainsthe potential to differentiate to derivatives of endoderm, mesoderm, andectoderm tissues throughout the culture, and (iii) is inhibited fromdifferentiation when cultured under proliferative conditions.

Furthermore, in a sixteenth aspect, the invention includes a cellularcomposition of adult stem cells which (i) will proliferate in an invitro culture for over one year, (ii) maintains a karyotype in which thechromosomes are euploid and not altered through prolonged culture, (iii)maintains the potential to differentiate to derivatives of endoderm,mesoderm, and ectoderm tissues throughout the culture, and (iv) isinhibited from differentiation when cultured under proliferativeconditions.

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

By “multipotential stem cell” is meant a cell that (i) has the potentialof differentiating into at least two cell types selected from a neuron,an astrocyte, and an oligodendrocyte, and (ii) exhibits self-renewal,meaning that at a cell division, at least one of the two daughter cellswill also be a stem cell. The non-stem cell progeny of a single MSC arecapable of differentiating into neurons, astrocytes, Schwann cells, andoligodendrocytes. Hence, the stem cell is “multipotent” because itsprogeny have multiple differentiative pathways. The MSC also has thepotential to differentiate as another non-neuronal cell type (e.g., askin cell, a hematopoietic cell, a smooth muscle cell, a cardiac musclecell, a skeletal muscle cell, a bone cell, a cartilage cell, apancreatic cell or an adipocyte).

By a “population of cells” is meant a collection of at least ten cells.Preferably, the population consists of at least twenty cells, morepreferably at least one hundred cells, and most preferably at least onethousand or even one million cells. Because the MSCs of the presentinvention exhibit a capacity for self-renewal, they can be expanded inculture to produce populations of even billions of cells.

By “substantially purified” is meant that the desired cells (e.g., MSCs)are enriched by at least 30%, more preferably by at least 50%, even morepreferably by at least 75%, and most preferably by at least 900% or even95%.

By “therapeutic protein” is meant a protein that improves or maintainsthe health of the cell expressing the protein or of a cell that is inproximity to the expressing cell. The term therapeutic protein shallencompass any protein that influences the proliferation,differentiation, and/or survival of the cells of the invention, withoutregard to the mechanism by which the therapeutic protein has thiseffect. Examples of therapeutic proteins include, without limitation,growth factors (NGF, BDNF, NT-3, NT4/5, HGF, TGF-β family members, PDGF,GDNF, FGF, EGF family members, IGF, insulin, BMPs, Wnts, hedgehogs, andheregulins) cytokines (LIF, CNTF, TNFμ interleukins, andgamma-interferon), and anti-apoptotic proteins (IAP proteins, Bcl-2proteins, Bcl-X_(L), Trk receptors, Akt, PI3 kinase, Gab, Mek, E1B55K,Raf, Ras, PKC, PLC, FRS2, rAPs/SH2B, and Np73). Additionally,therapeutic proteins include receptors for and the intracellularcomponents of signal transduction pathways. These signal transductionpathway are well known in the art (hedgehog pathway, Wnt pathway, BMPpathway, Notch pathway, FGF, etc), and one of skill will recognize thatexpression and/or treatment with components (ligands, receptors, orintracellular components) of a signal transduction pathway can modulatesignaling via that pathway with subsequent effects on cellproliferation, differentiation, and/or survival.

By “small molecule” is meant a compound having a molecular weight lessthan about 2500 amu, preferably less than about 2000 amu, even morepreferably less than about 1500 amu, still more preferably less thanabout 1000 amu, or most preferably less than about 750 amu. “Smallorganic molecule” are those small molecules which contain carbon.

By “plating conditions” is meant to include any and all parameters thatinfluence the proliferation, differentiation, and/or survival of cells.Plating conditions include, but are not limited to, changes in serumconcentration, changes in plating density, the use of various feederlayers and co-cultures, the addition of therapeutic proteins to theculture media, the addition of small molecules to the culture media, theaddition of pharmacological agents to the culture media, and theaddition of metals to the culture media. Any of these parameters may bealtered individually or in combination, and combinations of theseparameters can be manipulated at the same time or at different times.Additionally, it is understood, that the MSCs can be sorted prior toplating, such that a subpopulation of MSCs are subjected to thedifferentiation conditions. Sorting of the MSCs may be based on theexpression (or lack of expression) of a gene or protein. Furthermore,sorting of the MSCs may be based on cellular characteristics includingcell adhesion, or morphology.

By “peripheral tissue” is meant a tissue that is not derived fromneuroectoderm, for example peripheral tissue containing sensoryreceptors, and specifically includes olfactory epithelium, tongue, skin(including dermis and/or epidermis), and mucosal layers of the body(e.g., mouth, reproductive system).

By “epithelia” and “epithelium” in meant the cellular covering ofinternal and external body surfaces (cutaneous, mucous and serous),including the glands and other structures derived therefrom, e.g.,corneal, esophegeal, epidermal, and hair follicle epithelial cells.Other exemplary epithelial tissue includes: olfactory epithelium, whichis the pseudostratified epithelium lining the olfactory region of thenasal cavity, and containing the receptors for the sense of smell;glandular epithelium, which refers to epithelium composed of secretingcells; squamous epithelium, which refers to epithelium composed offlattened plate-like cells. The term epithelium can also refer totransitional epithelium, that which is characteristically found lininghollow organs that are subject to great mechanical change due tocontraction and distention, e.g. tissue which represents a transitionbetween stratified squamous and columnar epithelium. The term“epithelialization” refers to healing by the growth of epithelial tissueover a denuded surface.

By “skin” is meant the outer protective covering of the body, consistingof the corium and the epidermis, and is understood to include sweat andsebaceous glands, as well as hair follicle structures. Throughout thepresent application, the adjective “cutaneous” may be used, and shouldbe understood to refer generally to attributes of the skin, asappropriate to the context in which they are used.

By “epidermis” is meant the outermost and nonvascular layer of the skin,derived from the embryonic ectoderm, varying in thickness from 0.07-1.4mm. On the palmar and plantar surfaces it comprises, from withinoutward, five layers: basal layer composed of columnar cells arrangedperpendicularly; prickle-cell or spinous layer composed of flattenedpolyhedral cells with short processes or spines; granular layer composedof flattened granular cells; clear layer composed of several layers ofclear, transparent cells in which the nuclei are indistinct or absent;and horny layer composed of flattened, conified non-nucleated cells. Inthe epidermis of the general body surface, the clear layer is usuallyabsent. An “epidermoid” is a cell or tissue resembling the epidermis,but may also be used to refer to any tumor occurring in a noncutaneoussite and formed by inclusion of epidermal elements.

By “ectoderm” is meant the outermost of the three primitive germ layersof the embryo; from which are derived the epidermis and epidermaltissues such as the nails, hair and glands of the skin, the nervoussystem, external sense organs and mucous membrane of the mouth and anus.

By “mesoderm” is meant the middle of the three primitive germ layers ofthe embryo; from which are derived the heart, kidney, skeletal muscle,bone, cartilage, blood, endothelial lining of blood vessels, adiposetissue, and the urogenital system.

By “endoderm” is meant the innermost of the three primitive germ layersof the embryo; from which are derived the lungs, trachea, pharynx,thyroid, pharyngeal pouch derivatives, and the organs of the gutincluding the stomach, small intestines, large intestines, pancreas,liver, gall bladder, appendix, esophagus, rectum, anus, and urinarybladder.

By “differentiation” is meant the formation of cells expressing markersknown to be associated with cells that are more specialized and closerto becoming terminally differentiated cells incapable of furtherdivision or differentiation.

By “lineage committed cell” is meant a progenitor cell that is no longerpluripotent but has been induced to differentiate into a specific celltype, e.g., a dopaminergic neuron.

By “proliferation” is meant an increase in cell number.

By “non-adherent clusters” is meant that the cells of the invention areable to adhere to each other and form clusters which increase in size asthe cells proliferate, but these cells do not adhere to the substratumand grow in suspension, wherein the substratum is uncoated tissueculture plastic or a culture vessel coated with a neutral coating suchas agar or gelatin.

By “dissociating a sample” is meant to separate tissue into eithersingle cells, smaller cell clusters, or smaller pieces of tissue.

By “postnatal” is meant an animal that has been born at term.

By “a disease characterized by failure of a cell type” is meant one inwhich the disease phenotype is the result of loss of cells of that celltype or the loss of function of cells of that cell type.

By “autologous transplant” is meant that the transplanted material(e.g., MSCs or the progeny or differentiated cells thereof) is derivedfrom and transplanted to the same individual.

By “nucleic acid” is meant polynucleotides such as deoxyribonucleic acid(DNA), and, where appropriate, ribonucleic acid (RNA). The term shouldalso be understood to include, as equivalents, analogs of either RNA orDNA made from nucleotide analogs, and, as applicable to the embodimentbeing described, single (sense or antisense) and double-strandedpolynucleotides.

By “gene” is meant a nucleic acid comprising an open reading frameencoding a polypeptide, including both exon and (optionally) intronsequences.

By “transfection” is meant the introduction of a nucleic acid, e.g., anexpression vector, into a recipient cell by nucleic acid-mediated genetransfer.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked. It willalso be understood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring gene.

By “tissue-specific promoter” is meant a DNA sequence that serves as apromoter, i.e., regulates expression of a selected DNA sequence operablylinked to the promoter, and which effects expression of the selected DNAsequence in specific cells of a tissue, such as cells of neuronal orhematopoietic origin. The term also covers so-called “leaky” promoters,which regulate expression of a selected DNA primarily in one tissue, butcan cause at least low level expression in other tissues as well.

Other features and advantages of the present invention will becomeapparent from the following detailed description and the claims. It willbe understood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of example only, and various changes and modificationswithin the spirit and scope of the invention will become apparent tothose skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G are photographs showing that mouse skin-derived MSCs arenestin-positive and are capable of differentiating into neurons, glia,and smooth muscle cells.

FIG. 2 is a series of photographs showing that neonate and adult mouseskin-derived MSCs express both nestin (middle row) and fibronectinprotein (bottom row).

FIG. 3A is a series of photographs showing western blot analysis fornestin, neurofilament M (NF-M) and GFAP in cells differentiated fromneonate and adult mouse skin-derived MSCs.

FIG. 3B is a series of photographs showing that human skin-derived MSCsexpress nestin.

FIG. 3C is a series of photographs showing that a subset ofmorphologically complex cells expressed nestin and βtubulin, a profiletypical of newly-born neurons.

FIG. 3D is a series of photographs showing that GFP positive cells arealso positive for neuron-specific enolase.

FIG. 4A is a photograph showing the expression of A2B5, a marker foroligodendrocyte precursors, on undifferentiated mouse skin-derived MSCs.

FIG. 4B is a photograph showing the expression of the oligodendrocytemarker galactocerebroside (GalC) on cells differentiated from mouseskin-derived MSCs.

FIG. 5 is a series of photographs showing that the fate of mouseskin-derived MSCs can be manipulated by controlling plating conditions.

FIG. 6 is a series of photographs showing that neonate and adult mouseskin-derived MSCs can differentiate as adipocytes.

FIGS. 7A and 7B are photographs showing that nestin-positive,fibronectin-positive MSCs can be derived from mouse dermis.

FIGS. 8A and 8B are photographs showing that individual MSCs aremultipotent. Clones derived from single cells contained NF-M-positivecells (arrowheads) and CNPase-positive cells (arrows). Arrowheadsindicate cells that only express GFAP, while arrows indicate cellsexpressing both GFAP and CNPase.

FIGS. 9A and 9B are photographs of western blot analysis of cellsdifferentiated from mouse skin-derived MSCs (FIG. 9A) or of MSCsthemselves (FIG. 9B).

FIG. 10 is a series of photographs showing the effect of variouspharmacological agents on mouse skin-derived MSCs.

FIGS. 11A-11E are photographs of immunoprocessed sections of rat brainsinto which mouse skin-derived MSCs were transplanted.

FIG. 12 shows that nestin+, fibronectin+skin-derived MSCs isolated fromadult human scalp differentiate into cells that express a variety ofneural and non-neural markers, as measured by immunocytochemistry withantibodies to βIII-tubulin (A), CNPase (B), and smooth muscle actin (C),and GFAP (D).

FIG. 13 are photographs of skin-derived stem cells plated in 15% FBS inthe presence of skeletogenic supplements and cultured for two weeks. Thecells are stained with Alcian Blue which reveals nodules ofchondrocyte-associated acidic proteoglycans.

FIG. 14 are photographs of skin-derived stem cells plated in 15% FBS inthe presence of skeletogenic supplements and cultured for three weeks.The cells are stained with Alizarin Red which identifiedosteoblast-associated calcium accumulations.

FIG. 15 are photographs of skin-derived stem cells plated in 15% FBS inthe presence of skeletogenic supplements, cultured for three weeks, andco-stained with both Alcian Blue and Mizarin Red. Co-staining revealsthat the calcium deposits occur within a layer of chondrocyticproteoglycan accumulation.

FIG. 16 are photographs of skin-derived stem cells plated in 15% FBS inthe presence of skeletogenic supplements and cultured for 45 weeks, anddemonstrate the formation of optically dense deposits indicative of boneformation.

FIG. 17 shows that co-culture of GFP labeled skin-derived stem cellswith cardiac myocytes induces expression of fetal cardiac actin. Theexpression of fetal cardiac actin co-localizes with GFP indicating thatthe differentiated cell is derived from the skin-derived stem cell.

FIG. 18 shows that co-culture of GFP labeled skin-derived stem cellswith C2C12 cells induces expression of desmin. The expression of desminco-localizes with GFP, and the morphology of this desmin expressing cellis indicative of a skeletal muscle cell.

FIG. 19 shows RT-PCR analysis of skin-derived MSCs grown in spheres (S),plated in proliferation media for three days (3d), or plated inproliferation media for three days followed by two days in 5% serum(3d+2). The skin-derived MSCs express nestin, GATA-4, and Myf6. Positivecontrols (+ve) are: E10 brain (for nestin), embryoid bodies (forGATA-4), and muscle (for Myf6).

FIG. 20 shows that skin-derived MSCs express endodermal markers undercertain differentiation conditions. Skin-derived MSCs were culturedunder standard proliferation conditions in the presence or absence ofB-27 supplement. Differentiation was induced by plating cells in thepresence of nicotinamide, and the resulting differentiated cells wereanalyzed by quantitative RT-PCR. The graph demonstrates thatskin-derived MSCs differentiated in the presence of nicotinamide expressseveral markers of endodermal differentiation including GATA-4, HNF3α,Isl1, AFP, HNF3β, Ngn3, Pdx-1, and Insulin. Although cells proliferatedin either the presence or the absence of B27 supplement can be inducedto express endodermal markers, cells proliferated in B27 appear toexpress such markers to a higher degree.

FIG. 21 shows that agents, including therapeutic proteins and smallmolecules, influence the proliferation, differentiation, and/or survivalof skin-derived stem cells. Cells were dissociated and plated in thepresence of either 5% FBS, 5% FBS+retinoic acid (RA), or 5% FBS+BMP7.Cells were analyzed immunocytochemically for expression of neurofilamentM (NFM). Note the bottom panels shows a 40× magnification of the cells.

FIG. 22 shows that the skin-derived stem cells of the invention are acell population distinct from mesenchymal stem cells. Mesenchymal stemcells and skin-derived stem cells were cultured under identicalconditions, and immunocytochemical analysis was performed usingantibodies to nestin, fibronectin, vimentin, and cytokeratin. The toppanels are photographs of mesenchymal stem cells, and the bottom panelsare photographs of the skin-derived stem cells. Note not only thedifferences in protein expression, but also the differences inmorphology between the two cell types.

FIG. 23 shows that skin-derived stem cells isolated from human foreskinproliferate as non-adherent clusters in culture. The top panels showthat skin-derived stem cells specifically isolated from the dermal layerof human foreskin proliferate as non-adherent clusters. In contrast tohuman central nervous system derived stem cells, the survival andproliferation of human skin-derived stem cells is not dependent on LIF.The bottom panels show that skin-derived stem cells isolated fromforeskin express nestin and fibronectin.

FIG. 24 shows that skin-derived stem cells isolated from human foreskindifferentiate to form highly morphologically complex neurons as assayedby expression of bIII-tubulin and neurofilament-M (NF-M).

FIG. 25 shows that skin-derived stem cells isolated from human foreskindifferentiate to form glial cells as assayed by expression of GFAP andCNP.

FIG. 26 shows that skin-derived stem cells isolated from human foreskindifferentiate to form additional neuronal cells types as assayed byexpression of S100 and peripherin. S100 is a marker of bipolar cells andperipherin is a marker of peripheral neurons.

FIG. 27 shows that skin-derived stem cells isolated from human foreskindifferentiate to form non-neural cell types as assayed by expression ofsmooth muscle actin.

DETAILED DESCRIPTION OF THE INVENTION

We have substantially purified multipotent stem cells (MSCs) fromperipheral tissues of mammals, including skin, olfactory epithelium, andtongue. These cells proliferate in culture, so that large numbers ofstem cells can be generated. These cells can be induced todifferentiate, for example, into neurons, astrocytes, and/oroligodendrocytes by altering the culture conditions. They can also beinduced to differentiate into non-neural cells such as smooth musclecells, cartilage, bone, skeletal muscle, cardiac muscle, and adipocytes.The substantially purified neural stem cells are thus useful forgenerating cells for use, for example, in autologous transplants for thetreatment of degenerative disorders or trauma (e.g., spinal cordinjury). In one example, MSCs may be differentiated into dopaminergicneurons and implanted in the substantia nigra or striatum of aParkinson's disease patient. In a second example, the cells may be usedto generate oligodendrocytes for use in autologous transplants for thetreatment of multiple sclerosis. In a third example, the MSCs may beused to generate Schwann cells for treatment of spinal cord injury,cardiac cells for the treatment of heart disease, or pancreatic isletcells for the treatment of diabetes. In a fourth example, MSCs may beused to generate adipocytes for the treatment of anorexia or wastingassociated with many diseases including AIDS, cancer, and cancertreatments. In a fifth example, MSCs may be used to generate smoothmuscle cells to be used in vascular grafts. In a sixth example, MSCs maybe used to generate cartilage to be used to treat cartilage injuries anddegenerative conditions of cartilage. In still another example, MSCs maybe used to replace cells damaged or lost to bacterial or viralinfection, or those lost to traumatic injuries such as burns, fractures,and lacerations. If desired, in any of the foregoing examples, the cellsmay be genetically modified to express, for example, a growth factor, ananti-apoptotic protein, or another therapeutic protein. Similarly, theproliferation, differentiation, or survival of the MSCs of the inventioncan be influenced by modulating the cell culture conditions includingincreasing or decreasing the concentration of serum in the culturemedium and increasing or decreasing the plating density. Additionally,modulating the cell culture conditions includes contacting the MSCs (byadding to the culture medium) with an agent or agents that influenceproliferation, differentiation, or survival. Exemplary agents includetherapeutic proteins (i.e., growth factors, cytokines, cell-fatedetermining proteins, and anti-apoptotic factors), small molecules whichmay agonize or antagonize the effects of any of the foregoing proteins,and pharmacological agents. In one embodiment, the MSCs are presortedprior to plating and differentiation such that only a sub-population ofMSCs are subjected to the differentiation conditions. Presorting of theMSCs can be done based on expression (or lack of expression) of a geneor protein, or based on differential cellular properties includingadhesion and morphology.

The MSCs display some similarities to stem cells derived from mammalianforebrain, but also possess some distinctive differences. Firstly,non-adherent clusters of the proliferating MSCs of the invention aremorphologically distinct from CNS derived neurospheres. Additionally,when the MSCs of the present invention differentiate in the presence ofserum, about 5-20% of the differentiated cells express neuronal markers,whereas differentiated forebrain stem cells generate only a smallpercentage of neurons. Moreover, significant numbers of dopaminergicneurons are found in differentiated cultures of MSCs of the presentinvention, whereas such neurons have not been observed in cultures offorebrain stem cells differentiated in serum. Furthermore, we have notobserved any significant effects on the proliferation, differentiationor survival of the stem cells of the present invention when cultured inthe presence versus the absence of LIF. Thus, the MSCs of the inventionrepresent a novel stem cell population which can differentiate to formboth neural and non-neual cell types.

Cell Therapy

The multipotent stem cells of this invention may be used to preparepharmaceutical compositions that can be administered to humans oranimals for cell therapy. The cells may be undifferentiated ordifferentiated prior to administration. Dosages to be administereddepend on patient needs, on the desired effect, and on the chosen routeof administration.

The invention also features the use of the cells of this invention tointroduce therapeutic compounds into the diseased, damaged, orphysically abnormal CNS, PNS, or other tissue. The MSCs thus act as avector to transport the compound. In order to allow for expression ofthe therapeutic compound, suitable regulatory elements may be derivedfrom a variety of sources, and may be readily selected by one withordinary skill in the art. Examples of regulatory elements include atranscriptional promoter and enhancer or RNA polymerase bindingsequence, and a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the vector employed, othergenetic elements, such as selectable markers, may be incorporated intothe recombinant molecule. The recombinant molecule may be introducedinto the stem cells or the cells differentiated from the stem cellsusing in vitro delivery vehicles such as retroviral vectors, adenoviralvectors, DNA virus vectors and liposomes. They may also be introducedinto such cells in vivo using physical techniques such as microinjectionand electroporation or chemical methods such as incorporation of DNAinto liposomes. Such standard methods can be used to either transientlyor stably introduce heterologous recombinant molecules into the cells.The genetically altered cells may be encapsulated in microspheres andimplanted into or in proximity to the diseased or damaged tissue.

In one embodiment, the MSCs are used for the treatment of neurologicaldisease. In another aspect the MSCs of the present invention may also beused as a source of non-neural cells, for example adipocytes, bone,cartilage, and smooth muscle cells. As an example, PCT publicationWO99/16863 describes the differentiation of forebrain MSCs into cells ofthe hematopoietic cell lineage in vivo. The MSCs of the presentinvention appear to be more plastic and thus are highly likely to alsobe capable of differentiating into non-neural cell types, such ashematopoietic cells. Accordingly, the invention features methods oftreating a patient having any disease or disorder characterized by cellloss by administering MSCs of the present invention (or cells derivedfrom these cells) to that patient and allowing the cells todifferentiate to replace the cells lost in the disease or disorder. Forexample, transplantation of MSCs and their progeny provide analternative to bone marrow and hematopoietic stem cell transplantationto treat blood-related disorders. Other uses of the MSCs are describedin Ourednik et al. (Clin. Genet. 56:267-278, 1999), hereby incorporatedby reference. MSCs and their progeny provide, for example, cultures ofadipocytes and smooth muscle cells for study in vitro and fortransplantation. Adipocytes secrete a variety of growth factors that maybe desirable in treating cachexia, muscle wasting, and eating disorders.Smooth muscle cells may be, for example, incorporated into vasculargrafts, intestinal grafts, etc. Cartilage cells have numerous orthopedicapplications to treat cartilage injuries (i.e., sports injuries), aswell as degenerative diseases and osteoarthritis. The cartilage cellscan be used alone, or in combination with matrices well known in theart. Such matrices are used to mold the cartilage cells into requisiteshapes.

Transplantation and delivery of MSC's and their progeny may be at theactual site of cell damage or via the blood stream.

The following examples describe (i) the derivation of MSCs frompostnatal and adult mouse and rat tissue, (ii) the derivation of MSCsfrom human tissue, (iii) the differentiation of MSCs in vitro to bothectodermal and mesodermal derivatives, (iv) clonal analysisdemonstrating that single MSCs are multipotent, (v) the effects ofmodulating culture conditions on the proliferation, differentiation, andsurvival of MSCs, (vi) the transformation of MSCs with exogenous DNA,(vii) the in vivo differentiation of MSCs following transplantation.

Example 1 Purification of MSCs from Postnatal Mouse Olfactory Epithelium

MSCs from mouse olfactory epithelium were purified as described below.Postnatal mice were stunned with a blow to the head and thendecapitated. The heads were sagitally sectioned with a razor blade, andthe olfactory epithelia of about six postnatal (P1-P9) mouse pups werestripped from the conchae, nasal septum, and vomeronasal organs usingwatch-maker forceps. This tissue was placed into 3 mL of medium(DMEM/F-12 3:1) supplemented with 2% B-27 (Gibco, Burlington, Ontario,Canada), 20 ng/mL epidermal growth factor (EGF; Collaborative Research,Bedford, Mass.), 0.1% fungizone, and 0.5 mL/100 mLpenicillin/streptomycin (Gibco). Following collection, the epitheliawere teased apart with watchmaker forceps, releasing a large number ofsingle cells and small cell clusters. The cell suspension wastransferred to a 15 mL tube, and 7 mL of additional medium was added.The clusters were dissociated into single cells by manual titration witha 10 mL plastic pipette and passed through a 60 micron filter (Gibco).Typically, dissociated cells from the olfactory epithelia from six pupswere plated into two 50 mL tissue culture flasks and cultured in a 37°C., 5% CO₂ tissue culture incubator. Two days later, most cells in thecultures were dead or dying. A small number (less than 1% of the initialcell number) of large, phase bright cells were present, however, most ofwhich were attached to the flask bottom. Over the next two to six days,these cells divided and produced spherical clusters, which became largerover time. At four to five days in culture, there were approximately 500clusters of dividing cells per pup used in the original purification.Most of these cell clusters detached from the flask surface over thenext few days. These non-adherent cell clusters continued to grow andfused together to become macroscopic, reaching approximately 100 μm indiameter following 10 DIV. After 12 DIV, the non-adherent cell clustersbecame macroscopic, reaching approximately 200 μm or greater indiameter.

If EGF was not added to the medium, small clusters of dividing cellswere still seen by 4 DIV, indicating that the cells themselves wereproducing trophic factors in quantities that, in some cases, wassufficient to maintain their proliferation.

Greater than 95% of the cells in the dividing clusters expressed nestin,a marker for stem cells and neural stem cells. These nestin-positivecells could be repeatedly passaged, indicating that the cells were stemcells. Six days after purification, the medium (5 mL) was removed fromthe flasks. This medium contained many clusters of non-adherent stemcells that had detached from the flask surface. The detached cellsclusters were manually triturated with a fire-polished pipette, therebydissociating many of the cell clusters into single cells. The mediumcontaining the cells was then placed in a second flask with anadditional 15 mL of fresh medium (total volume=20 mL). After a furthersix days, one quarter of the medium was removed and the non-adherentclusters of cells were again triturated and transferred to a new flaskwith 15 mL fresh medium. These cells have been successfully passagedmore than twenty times without losing their multipotency.

Example 2 Differentiation of Mouse MSCs into Neurons, Astrocytes andOligodendrocytes

After the cellular clusters of Example 1 had been generated, they couldbe differentiated into neurons, astrocytes, and oligodendrocytes.Clusters from cultures 7 to 14 days after purification were plated ontopolylysine coated 35 mm culture dishes or 4 multiwell culture dishes, inDMEM/F12 media containing 2% fetal bovine serum (Hyclone, Logan, Utah)and 2% B-27 (containing no EGF). The medium was changed every three tofour days. Over the next six to nineteen days, cells migrated out of theclusters onto the dish surface. Some of these cells had the morphologyof neurons, astrocytes, or oligodendrocytes. We determined the phenotypeof these cells using the following antibodies: GFAP for astrocytes;neurofilament 160 (NF-160), MAP-2, βIII tubulin, and NeuN for neurons;and GC for oligodendrocytes. Antibodies to tyrosine hydroxylase (TH)were used to identify dopaminergic, noradrenergic, and adrenergicneurons. Dopamine-hydroxylase (DBH) was also used for noradrenergic andadrenergic neurons.

Astrocytes, neurons, and oligodendrocytes were all found todifferentiate from the MSCs of this invention, indicating that the cellswere multipotent. We also cultured MSCs from transgenic mice whichexpress β-galactosidase off of the neuron specific T1 α-tubulinpromoter, which allowed us to use staining with the ligand X-gal orantibodies for β-galactosidase as an additional neuronal marker. Weobserved β-galactosidase-positive cells.

Since the majority of differentiated cells remained in clusters, it wasnot possible to determine the percentage of cells expressing eachmarker. The majority of cells that migrated out of the clusters wereGFAP positive, while a large number of cells were either NeuN orβ-galactosidase positive. A lower number of cells were GC positive.Therefore the MSCs could differentiate into neurons, astrocytes andoligodendrocytes. TH-positive cells were also identified. TheseTH-positive cells are most likely dopaminergic neurons and notnoradrenergic or adrenergic neurons, since no cells were found to be DBHpositive. Significantly, no TH, GFAP or GC positive cells have ever beenreported in vivo in the nasal epithelium. Therefore the olfactoryepithelium-derived nestin-positive MSCs are capable of differentiatinginto cell types (e.g., oligodendrocytes, astrocytes, GABAergic neurons,and dopaminergic neurons) never found in the olfactory epithelium.

Like the originally-purified olfactory MSCs, MSCs passaged from two totwenty times could also differentiate into neurons, astrocytes, andoligodendrocytes. MSCs which had been passaged were plated onpolylysine-coated dishes. Cells migrated from the clusters and spreadout over the surface of the dish. After 16 DIV, cells that wereimmunopositive for GC, GFAP, βIII tubulin, NeuN, lacZ, or TH could beidentified. Moreover, the proportion of cells positive for the variousmarkers was similar to that seen in the differentiated cultures from theoriginal cultures.

Example 3 Purification of MSCs from Olfactory Epithelial Tissue of AdultMice and Rats

Similar to the foregoing results, MSCs were also purified from adultmouse and rat olfactory epithelium and vomeronasal organ using themethods described in Examples 1 and 2.

Adult mice and rats were anaesthetized with an overdose of somnitol, andthen decapitated. The olfactory and vomeronasal organ epithelia werestripped from the conchae and nasal septum and incubated in DMEM/F12medium for one to two days after their dissection and prior to the restof the purification procedure. After this incubation, the epithelia weredissociated in an identical manner as the epithelia from juvenile mice.Two days after the isolation, the majority of the cells were dead withthe exception of a very few large phase bright cells. These cellsdivided over the next few days, forming small clusters of dividing cellssimilar to those described in Example 1. These small clusters grew togive rise to the large clusters that detached from the culture dishsurface. After approximately six divisions, cells in some of theseclusters began to differentiate and spread out over the flask's surface,while some other clusters, which had been floating, reattached to thesurface and then produced differentiated cells. In some cases, cellsmultiplied to produce small clusters of cells, but did not grow to formlarge cell clusters like the postnatal cultures. We have passaged thesecells twenty times using the same procedure as that described above withrespect to the cells purified from juvenile olfactory epithelium. Theseproliferating cells from the adult were also nestin-positive.

After the cell clusters derived from adult tissue had been generated,the cells could be differentiated into neurons, astrocytes, andoligodendrocytes. Seven days after isolation, clusters were plated ontopolylysine-coated 35 mm culture dishes or multi-well culture dishes, inmedium containing 2% fetal bovine serum and 2% B-27, but no EGF. Overthe next month, cells migrated from the cell clusters and onto the dishsurface. We determined the phenotype of these cells using antibodies toastrocytes, neurons, dopaminergic neurons, and oligodendrocytes asdescribed above.

Neurons (including dopaminergic neurons), astrocytes, andoligodendrocytes were found, although the number of these cells was muchlower than the number obtained from the juvenile. The cells purifiedfrom adult olfactory epithelia are self-renewing and multipotent, andthus are MSCs.

Example 4 Purification of MSCs from Mouse Tongue

We derived MSCs from the tongue, another peripheral tissue that containssensory receptors. The tongue was dissected to remove the epitheliallayer that contains the sensory receptors and their underlying basalcells. This layer of tissue was triturated to produce single cells andthe single cells were plated in flasks containing DMEM/F12 mediasupplemented with B-27 and EGF, TGF, and/or bFGF, as described for theolfactory epithelium. After two to three days in a 37° C., 5% CO₂ tissueculture incubator, greater than 99% of the cells in the culture weredead or dying. A small number (less than 1%) of large phase-bright cellswere present, however, most of which attached to the flask bottom. Overthe next two to six days, these cells divided and produced sphericalclusters that became larger over time and detached from the flasksurface. The cells in these clusters were nestin-positive.

These nestin-positive MSCs can be passaged using the same techniques asused for the multipotent stem cells derived from the olfactoryepithelium. Similarly, the MSCs can be differentiated into neurons,astrocytes and oligodendrocytes using the techniques described herein.

Example 5 Purification of MSCs from Mouse Skin

Skin from neonatal mice aged 3-15 days was dissociated and cultured inuncoated flasks containing 20 mg/mL EGF and 40 mg/mL bFGF. Over thesubsequent one to five days, many (>90%) of the cells die. A smallpopulation of cells hypertrophy and proliferate to form small cellclusters growing in suspension. Some of these cells first attach to thetissue cluster plastic, hypertrophy and proliferate, and then detach asthe clusters become of sufficient size. Other cells never attach to thetissue culture plastic and instead proliferate in suspension from thebeginning. After four to five days, the cell clusters are small buteasily distinguishable as clusters of non-adherent, proliferating cells.By seven to ten days, many of the cell clusters reach diameters of asmuch as 100 μm, while by two weeks, the cell clusters are macroscopic ifleft unperturbed. Many cells adhered to the plastic, and many died, butby about three to seven days, suspended, non-adherent clusters of up toabout 20 cells formed. These suspended or floating cells weretransferred to a new flask seven days after initial culturing; again,many cells adhered, but the cells in the floating clusters proliferatedto generate larger clusters of more than about 100 cells (FIG. 1A, toppanel). These larger clusters were then isolated, dissociated andpassaged. By this process of selective adhesion, substantially purepopulations of floating clusters were obtained after 3 to 4 weeks. Cellsthat generated these clusters were relatively abundant; 1.5 to 2 cm² ofabdomen skin was sufficient to generate six 25 cm² flasks of floatingclusters over this period of time.

To determine whether clusters contained MSCs, we dissociated theclusters and plated the cells onto poly-D-lysine/laminin-coated dishesor chamber slides without growth factors and, 12 to 24 hours later,immunostained them for the presence of the neural precursor-specificmarker nestin. After three passages, the majority of the cells expressednestin (FIG. 1B, top panel), a property they maintained over subsequentpassages. They did not, however, express the p75 neurotrophin receptor,a marker for neural crest stem cells, as detected either byimmunocytochemistry or western blots. Additionally, they are negative,as detected by immunocytochemistry, for two proteins characteristic ofmesenchymal stem cells: vimentin and cytokeratin.

We also determined whether the skin-derived MSCs expressed fibronectin.Four lines of skin-derived MSCs cultured from either adult (FIG. 2; lefttwo columns) or neonatal (right two columns) mouse skin, cultured foreither long term (first and third columns) or short term (second andfourth columns) were each dissociated, plated for two days in DMEM/F12(3:1) containing 2% B-27 supplement, and then immunostained for nestinand fibronectin. As is demonstrated in FIG. 2, the majority of cellsexpressed both markers.

To determine whether clusters of cells could be generated from adults,skin of adult mice was dissociated and cultured as described above.Similar to neonatal mouse skin, most cells adhered to the flask or diedwhen first cultured. After three to seven days, however, clusters of upto approximately 20 cells were observed that subsequently increased insize. When these cells were passaged at least three times (FIG. 1A,bottom panel), and plated onto poly-D-lysine/laminin overnight in theabsence of growth factors, they too were immunopositive for nestin (FIG.1B, bottom panel) and fibronectin (FIG. 2). The nestin-positive cellsfrom adults and neonates have been passaged in this manner for over 30passages, during which time the number would have theoretically expandedat least 10⁹-fold (assuming a doubling time of approximately one week).

To determine whether these nestin-positive, fibronectin-positive cellsfrom skin could generate neural cell types, we analyzed neonatalskin-derived cells after three or more passages and greater by platingthem on poly-D-lysine/laminin in the absence of growth factors.Immunostaining (FIGS. 1C and 1D) and western blot analysis (FIG. 3A)revealed that the skin-derived cells expressed neuronal markers. Atseven days, a subpopulation of morphologically-complex cells coexpressednestin and neuron-specific βIII-tubulin, a profile typical of newly-bornneurons (FIG. 1C). At later time points of 7-21 days, cells alsoexpressed neurofilament-M (NF-M) (FIGS. 1D, 3A), neuron-specificenolase, and NeuN, three other neuron-specific proteins. Finally, someneurofilament-positive cells expressed GAD (FIG. 1D), a marker forGABAergic neurons, which are not found in the PNS. Similar results wereobtained for adult skin-derived MSCs, although at early passages some ofthe βIII-tubulin and neurofilament-positive cells were less typicallyneuronal in morphology.

Immunostaining and western blots revealed that both neonatal and adultMSCs generated cells expressing the glial markers GFAP and CNPase atseven to twenty-one days after plating (FIGS. 1D-1F, 2A).Double-labeling for these proteins demonstrated the presence of (i)cells that were GFAP-positive but not CNPase-positive (potentiallyastrocytes), (ii) cells that expressed CNPase but not GFAP (potentiallyoligodendrocytes or their precursors), and (iii) a small subpopulationthat were bipolar and expressed both CNPase and GFAP (potentiallySchwann cells) (FIG. 1E). A subpopulation of GFAP-positive cells alsoexpressed nestin, a finding previously reported for developing CNSastrocytes. Additionally, some cells were positive for A2B5, a markerfor oligodendrocyte precursors (FIG. 4). Like GAD-positive neurons,astrocytes and oligodendrocytes are normally found only in the CNS.

Double-labeling studies supported the following additional conclusions.First, glial versus neuronal markers were expressed in distinctsubpopulations of MSCs progeny. Second, after twenty passages,skin-derive MSCs were still able to differentiate into neurons and glialcells. Finally, skin-derived MSCs were able to generate smooth musclecells (as determined by both expression of smooth muscle actin (SMA) andmorphology; FIG. 1G), adipocytes (FIGS. 5 and 6), cartilage, bone,cardiac muscle, and skeletal muscle.

Example 6 MSCs Originate from the Dermal Layer of the Skin

The two major layers of the skin are the epidermis and the dermis. Todetermine the origin of the skin-derived MSCs, we dissected and culturedP7, P14, and P18 mouse epidermis and dermis. The two layers of the skinwere separated by incubating the skin pieces (1×2 cm²) in 0.2% trypsinat 40° C. for about 24-36 hours, or until the dermis could be separatedfrom the epidermis. The cells in each layer were dissociated separatelyand then cultured in DMEM/F12 (3:1) with B-27 supplement, EGF (20 ng/mL)and FGF (40 ng/mL). Only the cells derived from the dermis generatedclusters of cells similar to those derived from whole skin (FIG. 7A). Noviable cells were obtained from the epidermis. To characterize thedermis-derived cell clusters, the clusters were cultured for four weeksand then plated onto tissue culture chamber slides coated withpoly-D-lysine and laminin. After 24 hours, the cells were then processedfor immunocytochemistry. Like MSCs derived from whole mouse skin, thedermis-derived cells coexpressed nestin and fibronectin (FIG. 7B).

Example 7 Clonal Analysis Indicates that Skin-Derived MSCs areMultipotent

To determine whether skin-derived MSCs are multipotent, we isolatedsingle cells by limiting dilution of cells from clusters that threemonths prior had been derived from neonatal mice. We cultured the cellsfor five weeks in medium from the same culture line and containinggrowth factor, and then differentiated the cells for two weeks in mediumlacking growth factor but containing 3% rat serum. The cells were thenprocessed for immunocytochemistry. As is demonstrated in FIG. 8, singleclones of cells contained NF-M- and CNPase-positive cells (FIG. 8A), andGFAP- and CNPase-positive cells (FIG. 8B).

Example 8 Western Blot Analysis of Skin-Derived MSCs

For western blot analysis of skin-derived MSCs, four cultures (oneadult-derived line and three neonate-derived lines) that had beenpassaged from seven to 40 times were analyzed either as clusters orfollowing differentiation by plating in medium containing 1% FBS, B-27supplement, and fungizone for 14 days in 60 mm dishes coated withpoly-D-lysine and laminin. Cell lysates were prepared, and equal amounts(50-100 μg) of protein from each culture were separated on 7.5% or 10%polyacrylamide gels, transferred to membrane, and then probed withanti-nestin monoclonal antibody (1:1000; Chemicon), anti NF-Mpolyclonal) antibody (1:1000; Sigma), anti GFAP polyclonal antibody(1:1000, Dako), or anti fibronectin polyclonal antibody (1:1000; Sigma).As positive controls, we used cortical progenitor cells cultured in thepresence of CNTF (which results in astrocytic differentiation) or in theabsence of CNTF (which results in neuronal differentiation) and adultmouse cortex. As negative controls, we used sympathetic neurons andliver. As illustrated in FIG. 9A, western blotting confirmed theexpression of GFAP and NF-M in cultures differentiated from both adultand neonate skin-derived MSCs. Similarly, FIG. 9B illustrates theexpression of both nestin and fibronectin in adult and neonateskin-derived MSC clusters.

Example 9 MSC Differentiation can be Modulated by Plating Conditions

As is illustrated above, when clusters of skin-derived MSCs aredissociated and plated in medium containing FGF and EGF, most of thenestin-positive cells become neurofilament-positive. We have found thatwhen the cells are plated in medium containing 10% FBS, the cells adopta morphology similar to that displayed by adipocytes. The adoption ofthe adipocyte cell fate was confirmed by staining with Oil Red O (FIG.5). The ability of 10% FBS to induce adipocyte differentiation was truefor both adult and neonate skin-derived MSCs (FIG. 6).

This example demonstrates both the ability of skin-derived MSCs todifferentiate to mesodermal cell types, and the significant effects ofplating conditions on the proliferation, differentiation and survival ofskin-derived MSCs. In addition to serum concentration, other platingconditions can be altered to influence the proliferation,differentiation, and survival of these cells. Such plating conditionsinclude plating density, the addition of pharmacological agents to theculture media (i.e., pharmacological inhibitors), the addition oftherapeutic protein(s) to the culture media (i.e., growth factors,cytokines, anti-apoptotic proteins), and the addition of small moleculesthat agonize or antagonize the function of a protein(s) and/or modulatesignaling through a signal transduction pathway important in regulatingthe proliferation, differentiation, or survival of the skin-derivedMSCs. These parameters can be altered individually, or in combination toinfluence the proliferation, differentiation or survival of theskin-derived MSCs. For example, one or more therapeutic proteins can beadded to the culture media. Furthermore, therapeutic proteins can beadded in combination with changes in plating density. Still anotherembodiment combines the addition of therapeutic protein(s) with theaddition of a small molecule. Additional plating conditions include theco-culture of the skin-derived MSCs with other cells or cell types, andthe pre-sorting of the skin-derived MSCs prior to plating.

Examples of the effects of altering several different plating conditionsare presented below.

Example 10 Pharmacological Inhibitors Affect Survival and Proliferationof Skin-Derived MSCs

When skin-derived MSCs are plated for three days in proliferation mediumcontaining FGF, they typically exhibit a spherical morphologycharacteristic of their proliferative state (FIG. 10). We tested theability of pharmacological agents to alter this phenotype. Supplementingthe medium with PD098059 (an inhibitor of the ERK MAPK pathway) causedproliferating cells to flatten and differentiate (FIG. 10), whilesupplementing with LY294002 (an inhibitor of the PI-3-K pathway), causedthe cells to die (FIG. 10). The p38 MAPK inhibitor SB203580 had noobserved effect on the proliferating skin-derived MSCs.

These and other pharmacological agents could be added to the culturemedia to influence cell proliferation, differentiation, and survival.Pharmacological agents can be added alone or in combination, andcombinations of agents can be co-administered or administered atdifferent times. Additionally, pharmacological agents can beadministered in combination with one or more therapeutic proteins orsmall molecules to influence cell proliferation, differentiation, andsurvival. Such combinations of pharmacological agents and therapeuticproteins can be co-administered or administered at different times toinfluence cell proliferation, differentiation, and survival.

Example 11 Purification of Nestin-Positive Cells from Adult Human Skin

We have purified nestin-positive cells from human scalp. To purify MSCsfrom human skin, we utilized tags of scalp tissue generated by placementof a stereotactic apparatus during neurosurgery. Scalp tags totaling 1cm² or less from each of eight individuals were used. The skin includeddermal and epidermal tissue. Tissue was cut into smaller pieces thatwere then transferred into HBSS containing 0.1% trypsin for fortyminutes at 37° C. Following trypsinization, tissue samples were washedtwice with HBSS and once with DMEM:F12 (3:1) supplemented with 10% ratserum to inactivate the trypsin. Trypsinized tissue was thenmechanically dissociated by trituration in a pipette and the resultingdispersed cell suspension was poured through a 40 μm cell strainer intoa 15 mL tube. The tube was then centrifuged for five minutes at 1000 rpm(˜1200×g). The cells were resuspended in DMEM:F12 medium containing 40ng/mL bFGF, 20 ng/mL EGF, 2% B-27 supplement, and antibacterial andantifungal agents, and then cultured in 12 well plastic tissue cultureplates. Every seven days, the cell clusters are harvested bycentrifugation, triturated with a fire-polished pasteur pipette, andcultured in fresh medium.

As for the use of rodent skin, most cells (>75%) adhered to the plasticor died, but after seven days, small floating clusters of cells wereobserved. These clusters were then partially dissociated and transferredto new wells, where they slowly increased in size. After additionalpassaging, clusters were plated on poly-D-lysine/laminin in 3% FBS withno growth factors, and analyzed for the presence of neural markers.

Within two weeks, greater than 30% of the cells within the cell clusterswere nestin-positive. Immunolabeling of four to six week old culturesalso revealed that many of the cells in the clusters werenestin-positive with the percentage varying from less than 50% togreater than 80% two to three days after plating, and that greater than70% of the cells were fibronectin positive. Double-labelimmunocytochemistry at the same or longer time-points revealed that, inall cultures, some nestin-positive cells also expressed βIII-tubulin anddisplayed elongated neurites. Thus, adult human skin is a source fornestin-positive and fibronectin positive MSCs cells that, whendifferentiated, can express neuron-specific proteins.

Example 12 Purification and Differentiation of MSCs Derived from OtherHuman Peripheral Tissues Containing Sensory Receptors

MSCs can be purified from human olfactory epithelium using the sameprocedures as described for the purification of stem cells from rodentolfactory epithelium. Source material is acquired by surgical removal ofolfactory epithelial tissue from the donor. Because the MSCs are capableof proliferation and self-renewal, little source tissue is required.Preferably, the amount is at least about 1 mm³. Conditions for culturinghuman cells are described in Example 11, above. Other conditions areknown to those skilled in the art, and can be optimized forproliferation or differentiation of neural stem cells, if desired.

We can purify MSCs from other peripheral tissues containing sensoryreceptors, other than the olfactory epithelium, tongue, and skin, usingtechniques described herein. Passaging and differentiation of thesecells is also performed using the same techniques described herein.Other peripheral tissues containing sensory receptors include, forexample, mucosal membranes from the mouth or reproductive system.

Example 13 Transformation of MSCs

In therapy for neurodegenerative diseases, it may be desirable totransplant cells that are genetically modified to survive the insultsthat caused the original neurons to die. In addition, MSCs may be usedto deliver therapeutic proteins into the brain of patients withneurodegenerative disorders to prevent death of host cells. Exemplarytherapeutic proteins are described herein. In still another example,MSCs can be induced to differentiate into a desired cell type bytransfecting the cells with nucleic acid molecules encoding proteinsthat regulate cell fate decisions (e.g., transcription factors such asIsl-1, en-1, en-2 and nurr-1, implicated in regulating motorneuron andstriatal phenotypes). Using such a method, it is possible to induce thedifferentiation of the specific cell types required for transplanttherapy. Therefore, it would be advantageous to transfect MSCs withnucleic acid molecules encoding desired proteins. We have previouslyused recombinant adenovirus to manipulate both postmitotic sympatheticneurons and cortical progenitor cells, with no cytotoxic effects. We nowhave established that olfactory epithelial-derived MSCs and skin-derivedMSCs can each be successfully transfected with high efficiency and lowtoxicity. MSCs can be transfected either transiently or stably using notonly adenoviral mediated methods, but also using lipofectamine orelectroporation.

Example 14 Differentiation of MSCs into the Appropriate Cell Type InVivo Following Transplantation into Adult Rodent Brain

One therapeutic use for the MSCs of the present invention is autologoustransplantation into the injured or degenerating CNS or PNS to replacelost cell types and/or to express therapeutic molecules. We demonstratebelow that the MSCs can differentiate into neurons when transplantedinto the adult brain.

If desired, the dopaminergic innervation of the adult striatum can beunilaterally destroyed by a local infusion of 6-hydroxydopamine underconditions in which noradrenergic neurons are spared. Several weekslater, MSCs are transplanted into both the intact and lesioned striatum.Alternatively, the cells can be transplanted into unlesioned animals.The fate of the transplanted MSCs is then determined byimmunohistochemistry. Exemplary transplantation studies are describedbelow. These studies demonstrate that transplanted MSCs candifferentiate into neurons in vivo, as they can in vitro. In the formercase, differentiation and cell fate choice is controlled by the localenvironment into which each cell is placed. Both in vitro-differentiatedand undifferentiated cells are useful therapeutically in the treatment,for example, of neurodegenerative disease (e.g., Parkinson's disease andmultiple sclerosis) or spinal cord injury. For example, dopamingergicneurons differentiated from MSCs, or the MSCs themselves, may betransplanted into the substantia nigra or the striatum of patientshaving Parkinson's disease. If desired, the MSCs may also begenetically-modified to express a desired protein. Such geneticmodification may help influence the proliferation, differentiation, andsurvival of the MSCs. In one embodiment, the genetic modificationprotects the transplanted cells from the conditions which caused thedegeneration of the endogenous cells.

In one example, the dopaminergic innervation to adult rat striatum wasfirst unilaterally lesioned with the chemotoxin 6-hydroxydopamine, andthe efficacy of the lesions was tested two weeks later byamphetamine-induced rotational behavior. Two days prior totransplantation, rats were immunosuppressed with cyclosporin. MSCs,produced from olfactory epithelia as described herein, were thenstereotactically injected into the caudate-putamen complex on both thelesioned and unlesioned sides. Sixteen days following transplantation,animals were sacrificed, and sections of the striatum were analyzed fornestin- and TH-immunoreactivity. Five of eight animals receivedsuccessful injections of MSCs in the striatum. Of these, four animalsshowed evidence of a nestin-positive tract on both the lesioned andunlesioned sides, although tracts on the lesioned side appeared to bemore intensely nestin-immunoreactive. On adjacent sections, TH-positivecells were observed confined to an area close to the transplant tract onboth the lesioned and unlesioned side. As many as 25-30 TH-positivecells were identified on each section. Cell morphology varied fromsmall, round cells lacking processes to neurons that weremorphologically complex with multiple fine processes. In some cases, theprocesses of these TH-positive neurons extended into the striatum fordistances of up to 300 μm.

To confirm that these TH-positive neurons derived from the MSCs, weperformed two sets of experiments in which the transplanted cells weredetectably-labeled. In one set of experiments, transplanted MSCs werederived from T1:nlacZ transgenic mice, in which the neuron-specific T1α-tubulin promoter drives expression of a nuclear-localizedβ-galactosidase marker gene. Immunohistochemical analysis of animalsreceiving the transgenic MSCs revealed the presence ofβ-galactosidase-positive neurons within the transplant tract, confirmingthat the transplanted MSCs generated neurons in vivo, as they did invitro. In a second set of experiments, MSCs were labelled with BrdU for18 hours, washed to remove the BrdU label, and then transplantedunilaterally into the 6-hydroxydopamine-lesioned striatum of animals (10rats, 4 mice) prepared as described herein. Immunohistochemical analysiswith an anti-BrdU antibody revealed that all animals showed evidence ofBrdU-positive transplant tracts. Immunocytochemistry with anti-GFAPrevealed that, in both xenografts and allografts, GFAP-positive cellswith heterogeneous morphology were concentrated at the transplant site,but were also present in moderate amounts over the entire ipsilateralhemisphere, with additional scattered reactive astrocytes seen in thecontralateral hemisphere. GFAP-BrdU double-labelled cells were presentmainly within or close to the transplant tract, and varied in morphologyfrom small, round cells with only a few processes, to large polygonal orfusiform cells with multiple processes. Immunohistochemistry withanti-TH revealed that TH-BrdU double-labeled cells were also present,although these were few in number relative to GFAP-BrdU positive cells.BrdU-TH double-labeled cells were mainly small to medium-sized withoutprocesses, although some examples of double-labeled cells with processeswere found within and adjacent to, the transplant tract. Thus, MSCsgenerated astrocytes and neurons in vivo, and a subpopulation of thelatter were TH-positive. Together, these findings show that peripheraltissue-derived MSCs are capable of generating cell types that are neverfound within olfactory tissue, including oligodendrocytes andTH-positive neurons.

To determine whether skin-derived MSCs also generate differentiatedneural cell types in vivo, we tagged adult mouse skin-derived MSCs with(i) BrdU, and (ii) a recombinant adenovirus expressing GFP, and thentransplanted them as cell clusters of about 20 to about 100 cells intothe lateral ventricles of P2 rats. Immunostaining fourteen days laterrevealed that, in all animals analyzed (n=8), transplanted cells hadmigrated extensively (FIG. 11A). In particular, tagged cells hadintegrated into the cortex, the hypothalamus and the amygdala in all,and into the hippocampus in two of the transplanted brains (FIG. 11A).In the cortex, GFP-positive cells were located in patches (FIGS. 11A,11B) or occasionally as single cells (FIG. 11C), including some that hadintegrated into and adopted the morphology of layer V pyramidal neurons(FIGS. 11B, 11C). These cells had triangular-shaped soma, and projecteda presumptive apical dendrite from layer V towards layer I, in a mannersimilar to the endogenous layer V neurons. That these cells were neuronswas demonstrated by double-labeling for neuron-specific enolase (FIG.3D). Immunocytochemical analysis also confirmed that these weretransplanted cells, as BrdU-positive cells were present in the samelocations as GFP-positive cells in all brains (FIG. 11B).

In both the amygdala and hippocampus, transplanted cells also displayedneuronal morphology. In the amygdala, GFP and BrdU-positive cells werelarge, with prominent nuclei, and extensive processes (FIG. 11E). In thehippocampus, transplanted cells had integrated into both the dentategyrus and pyramidal cell layers, and their morphology was typical of theendogenous granule and pyramidal cells, respectively (FIGS. 11A, 11E).GFP-positive staining was also seen within the molecular layer. Finally,GFP- and BrdU-positive cells were observed in other locations, such asthe hypothalamus, where the morphology of many cells was not typicallyneuronal.

Skin-derived MSCs transplanted into adult rats also survive andintegrate. We labeled adult mouse skin-derived MSCs that had beenpassaged more than thirty times with the nuclear dye 33258, washedextensively, and then injected the cells stereotactically into thebrains of adult rats that were immunosuppressed with cyclosporin. Fourweeks later, we sacrificed the animals by perfusion and processed thebrains for histological examination. Hoeschst-labeled cells were presentin the hippocampus, olfactory bulb, and striatum. From these data, weconclude that the transplanted skin-derived MSCs are capable of survivalfollowing transplantation. Moreover, cells are capable of migrating fromthe site of injection to numerous brain regions.

Skin-derived MSCs are also capable of survival, migration, andintegration following transplantation into a hemisected adult mousespinal cord. In this example, the cells were injected into the injuredsides of hemisected spinal cords. Eight days later, the animals weresacrificed and the spinal cords processed for histological analysis.Hoechst-labeled cells were present at the site of the initial injection,and had also migrated extensively into the injured spinal cord.

Example 15 Differentiation of Non-Neural Cells from MSCs

In addition to being capable of differentiating as neural cells (i.e.,neurons, oligodendrocytes, astrocytes, and Schwann cells), theperipheral tissue-derived MSCs are capable of differentiating asnon-neural cells that are normally not found in the tissue from whichthe cells were derived. For example, we have demonstrated that theskin-derived MSCs can differentiate as smooth muscle cells, cartilage,bone, muscle, and adipocytes. It is likely that the cells describedherein have even greater potential. Conditions for the differentiationof the MSCs into smooth muscle cells, adipocytes, cartilage, bone,skeletal muscle, and cardiac muscle are described herein. Additionally,we show that the skin-derived MSCs can express RNA transcriptsconsistent with endodermal differentiation. These findings demonstratethat the skin-derived MSCs have potential to differentiate along allthree germ layers.

Signals or conditions sufficient for inducing MSCs to differentiate asother cell types (e.g., lymphocytes, cardiac muscle cells, skeletalmuscle cells, melanocytes, and pancreatic cells) are known in the art.For example, unique signals induce neural crest-derived stem cells tobecome melanocytes, cartilage, smooth muscle cells, or bone (for review,see LaBonne and Bronner-Fraser, J. Neurobiol., 36:175-189, 1998;Sieber-Blum, Intl. Rev. Cytol. 197:1-33, 2000). Conditions for inducingCNS-derived neural stem cells to differentiate as non-neural cells suchas smooth muscle cells, skeletal muscle cells, hepatocytes,hematopoietic cells, osteocytes, and chondrocytes have similarly beenelucidated (Bjornson et al., Science 283:534-537, 1999; Tsai and McKay,J. Neurosci. 20:3725-3735, 2000; Keirstead et al., J. Neurosci.19:7529-7536, 1999; Mujtaba et al., Dev. Biol. 200:1-15, 1998; Clark etal., Science 288:1660-1663, 2000).

Our recent discovery that MSCs maintain the potential to produce bothneural and non-neural cell types has been accompanied by the discoverythat non-neural stem cells such as bone marrow-derived stem cells (i.e.,stromal cells or mesenchymal stem cells) also have the potential toproduce a wide variety of neural and non-neural stem cells (Ferrari etal., Science 279:1528-1530, 1998; Gussoni et al., Nature 401:390-394,1999; Peterson et al., Science 284:1168-1170, 1999; Pereira et al.,Proc. Natl. Acad. Sci. USA 92:4857-4861, 1995; Prockop, Science276:71-74, 1997; Kessler and Byrne, Annu. Rev. Physiol. 61:219-242,1999; Pittenger et al., Science 284:143-147). The peripheraltissue-derived MSCs described herein can be induced to differentiateinto both neural and non-neural cells that are not normally found in thetissue from which the MSCs were derived.

-   -   a. MSCs can differentiate to smooth muscle: For induction of        differentiation into smooth muscle cells, the cell clusters were        centrifuged, the growth factor-containing supernatant removed,        and the clusters resuspended in fresh media containing B-27        supplement and either 3% rat serum or 1-3% fetal bovine serum.        The clusters were then plated onto dishes coated with        poly-D-lysine/laminin, and the medium was changed every 3 to 7        days. Smooth muscle cells were identified by immunocytochemistry        with an antibody to smooth muscle actin (SMA).    -   b. MSCs can differentiate to adipocytes: For induction of        differentiation into adipocytes, the cell clusters were        centrifuged, the growth factor-containing supernatant removed,        and the clusters resuspended in fresh media containing B-27        supplement with 10% fetal bovine serum. The clusters were plated        onto dishes coated with D-lysine/laminin. Differentiated        adipocytes were identified by OilRed staining.    -   c. MSCs can differentiate to a skeletogenic fate: For induction        of differentiation along a skeletogenic lineage, the cell        clusters were centrifuged, the growth factor containing        supernatant removed, and the clusters resuspended in fresh media        containing B-27 supplement with 15% fetal bovine serum including        skeletogenic supplements. The skeletogenic supplement includes        dexamethasone (100 nM), ascorbic acid (50 nM), and        b-glycerophosphate (10 mM). After 2 weeks, Alcian Blue staining        of the cultures reveals nodules of staining characteristic of        chondrocytes. Alcian Blue staining indicates that the        chondryocytes produce acidic proteoglycans (FIG. 13). After 3        weeks, calcium accumulation is observed in the cultures        indicative of osteoblast activity. The calcium accumulation is        assayed by Alizarin Red S staining (FIG. 14). Alcian        Blue/Alizarin Red co-staining at 3 weeks demonstrates that the        calcium accumulation occurs within a layer of chondrocytic        proteoglycans (FIG. 15). Finally, by about 4-5 weeks, optically        dense deposits, indicative of bone formation, are observed in        the culture (FIG. 16).    -   d. MSCs can give rise to muscle: To assess the ability of the        skin-derived stem cells of the invention to differentiate along        a muscle lineage, we co-cultured GFP-labelled skin-derived stem        cells with either cardiac myocytes or with C2C12 skeletal        myoblasts. After several days of co-culture the skin-derived        stem cells were analyzed based on both morphology and on protein        expression. Cells co-cultured with cardiac myocytes express        fetal cardiac actin, and the fetal cardiac actin expression        co-localizes with GFP (indicating that the expressing cells are        derived from the skin-derived precursors) (FIG. 17). Fetal        cardiac actin is expressed in both cardiac and skeletal muscle,        and the morphology of these cells is consistent with either two        cardiac muscle cells or with a single multinucleated skeletal        myotube. However these results indicate that skin-derived stem        cells can differentiate to a muscle cell type. Cells co-cultured        with C2C12 cells give rise to desmin positive cells, and desmin        expression co-localizes with GFP (FIG. 18). The morphology and        protein expression of the skin-derived stem cells cultured in        this manner is consistent with their differentiation to skeletal        muscle. These experiments indicate that skin-derived stem cells        can differentiate to produce skeletal muscle, and likely can        also differentiate to produce cardiac muscle.    -   e. MSCs can express endodermal markers: We have shown that the        skin-derived MSCs of the invention can differentiate to give        rise to both neural and non-neural cells. We have presented six        examples of mesodermal cell types that arise from        differentiation of the MSCs. We now present evidence that the        MSCs can also express transcripts consistent with endoderm        differentiation. FIG. 19 shows RT-PCR analysis demonstrating        that MSCs express the endodermal marker GATA-4. In a second        experiment, skin-derived MSCs were cultured under standard        proliferation conditions in the presence or the absence of B-27        supplement Cells were dissociated and plated in media        supplemented with nicotinamide. Differentiated cells were        analyzed by RT-PCR for the expression of several endodermal        markers including GATA-4, HNF3α, Isl1, AFP, HNF3β, Ngn3, Pdx-1,        and Insulin. FIG. 20 summarizes the results of this experiment        which demonstrates that cells differentiated in the presence of        nicotinamide express markers of endodermal differentiation.        Additionally although endodermal differentiation is observed in        cells that were proliferated in either the presence or the        absence of B27 supplement, the cells proliferated in the        presence of B27 expressed higher levels of endodermal markers        than cells proliferated in the absence of B27. This data        demonstrates that the skin-derived MSCs of the invention can        differentiate to cell types derived from all three germ layers.        Additionally, these experiments demonstrate that the modulating        of multiple plating conditions (in this case the addition of        both B27 supplement and nicotinamide), at different times, can        effect the differentiation of the skin-derived MSCs.

Skin-derived MSCs can differentiate to cell types of both neural andnon-neural lineages. We demonstrate that the MSCs can give rise toseveral different non-neuronal cell types including smooth muscle cells,adipocytes, cartilage, bone, skeletal muscle, and cardiac muscle.Additionally, we show that the skin-derived MSCs can express transcriptsconsistent with endoderm differentiation. The tremendous differentiativepotential of skin-derived MSCs suggests that in addition to the manycell types shown here, MSCs can also give rise to other mesodermal andendodermal cell types. Furthermore, these results demonstrate thatchanges in plating conditions (i.e., alterations in serumconcentrations, the addition of pharmacological agents and smallmolecules, and/or co-culturing cells with other cell types) can havedramatic effects on cell proliferation, differentiation and/or survival.

Example 16 Contacting MSCs with Agents to Influence Differentiation

As described in detail above, the proliferation, differentiation, orsurvival of the cells of the invention can be influenced by modulatingthe culture conditions. For example, we have shown that changes in theplating conditions, or the addition of pharmacological agents to theculture medium influences the proliferation, differentiation and/orsurvival of MSCs.

We show that the proliferation, differentiation or survival of MSCs canalso be influenced by contacting the cells with a therapeutic proteinincluding one or more cytokine, growth factor, extracellular protein,etc. One of skill will recognize that the concentration of these agentscan be altered to determine the optimal dose. Additionally, thetherapeutic proteins may be added alone, or in combination, andcombinations of proteins may be administered simultaneously or atvarying timepoints.

Skin derived MSCs were obtained and cultured as described in detailabove. To induce differentiation, cells were plated in the presence of5% serum supplemented with either retinoic acid or BMP-7. Neuronaldifferentiation was analyzed using a polyclonal anti-neurofilamentantibody. Addition of either retinoic acid or BMP-7 enhances the numberand complexity of neurofilament positive cells, in comparison to cellsdifferentiated in the presence of serum alone (FIG. 21).

Example 17 Skin-Derived MSCs are a Distinct Population of Stem Cells

We have demonstrated that the skin-derived multipotent stem cells of theinvention can differentiate to produce both neural and non-neural celltypes. Furthermore, we have demonstrated that these skin-derived stemcells can produce at least six mesodermal cell types (smooth muscle,adipocyte, cartilage, bone, skeletal muscle, and cardiac muscle). Theability of the stem cells of the invention to differentiate alongmesodermal lineages is a characteristic of mesenchymal stem cellspreviously isolated from sources including bone marrow.

Although previous experiments using mesenchymal stem cells indicate thatsuch cells are selectively adherent (in contrast to the skin-derivedcells of the invention), we performed morphological andimmunocytochemical analysis to demonstrate that the skin-derived cellsof the invention are distinct from the mesenchymal stem cells previouslyidentified. Bone marrow derived mesenchymal stem cells were obtainedfrom BioWhittaker, and were cultured under the conditions describedherein for skin-derived stem cells.

When grown under identical conditions, the two cell populations havesignificantly different morphology and growth characteristics. Themesenchymal stem cells do not proliferate in suspension when culturedunder conditions which allow the skin-derived stem cells to grow asnon-adherent clusters as described in detail herein. The cells weredissociated and plated overnight under the conditions described for theskin-derived cells. The two cell types are morphologically distinct: theskin-derived cells are considerably smaller while the mesenchymal stemcells have a more flattened appearance. Additionally althoughmesenchymal stem cells rapidly proliferate in standard mesenchymal cellmedium, they survive but do not readily proliferate under the conditionsused here.

Immunocytochemical analysis further illustrates the differences betweenthese two cell types. Cells were dissociated, plated overnight, andanalyzed immunocytochemically for the expression of vimentin,cytokeratin, nestin, and fibronectin. Both mesenchymal stem cells andthe skin-derived multipotent stem cells of the invention expressfibronectin. However, the two cell types differed in the expression ofthe other three markers analyzed. Skin-derived stem cells express nestin(mesenchymal stem cells do not). Mesenchymal stem cells expressvimentin, and a sub-set of the mesenchymal stem cells in culture expresscytokeratin (skin-derived stem cells express neither vimentin norcytokeratin) (FIG. 22).

Despite the ability of skin-derived stem cells to differentiate alongseveral mesodermal lineages, the stem cells of the invention aredistinct from previously identified mesenchymal stem cells. Thesedifferences are demonstrated by the differential morphology and proteinexpression observed when mesenchymal stem cells are cultured under theconditions described herein for the proliferation and differentiation ofskin-derived stem cells.

Example 18 Isolation of Skin-Derived Multipotent Stem Cells from HumanForeskin

We have previously demonstrated that the multipotent stem cells of theinvention can be isolated from both rodent and human skin. Exemplarysamples have been obtained from the scalp, back, and abdomen of donors.One of the unique advantages of the present invention is that skinrepresents a plentiful and easily accessible source of autologous orheterologous stem cells for transplantation.

However, in addition to autologous or heterologous skin samples takenspecifically to generate stem cells for transplantation, skin samplesare routinely harvested from healthy donors in the course of manymedical procedures. Such samples represent a plentiful source of tissuefor the generation of skin-derived stem cells. Such stem cells, or thedifferentiated progeny thereof, could be used for research purposes, aswell as for autologous or heterologous transplantation. Exemplaryprocedures which generate excess skin include circumcision and cosmeticsurgery (e.g., face lifts, liposuction, and “tummy tucks”). Typically,the excess tissue generated following these procedures is removed anddiscarded from otherwise healthy patients.

In one embodiment, stem cells can be isolated and cultured from saidexcess tissue. Such stem cells, or the differentiated progeny thereof,can be used immediately for transplantation or research purposes.Alternatively, the stem cells, or the differentiated progeny thereof,can be banked for later use by the donor (autologous transplantation),or for the treatment of a related or unrelated recipient.

In another embodiment of the present invention, stem cells are harvestedfrom the foreskin of a male patient. The stem cells, or thedifferentiated progeny thereof, can be stored for later use by eitherthe same male patient or his blood relatives. Alternatively, the stemcells, or the differentiated progeny thereof, can be used for thetreatment of an unrelated recipient.

Foreskin samples from human patients were obtained from surgeonsperforming circumcisions. The samples were taken from males ranging inage from newborns to adolescents. We note that we have generatedproliferating cultures of non-adherent skin-derived stem cells from 21samples, and have not observed any significant differences in thesurvival, proliferation, or differentiation characteristics among thecultures based on the age of the donor.

The quantity of tissue obtained following circumcision is relativelysmall. Accordingly, we reasoned that the number of stem cells in saidtissue may be relatively low, and that the survival of cultures derivedfrom these samples may improve if the stem cells could be enriched inrelation to non-stem cells in the sample. Previous studies demonstratedthat skin-derived stem cells reside in the dermal layer of the skin.Thus, we employed a novel method for isolating and culturing stem cellsfrom limiting quantities of tissue. Briefly, foreskin samples were firstcut into pieces and then enzymatically digested to separate the dermaland epidermal layers. Specifically, we digested the tissue for 24-48hours at 4° C. in an enzyme blend containing collagenase and eitherDispase or thermolysin. However, one of skill in the art can readilyselect from among commercially available proteases to choose one or moreenzymes which would achieve a similar effect. Following separation ofthe dermal and epidermal layer, the dermal layer was dissociated byfurther digestion in the enzyme blend for 30 minutes at 37° C. followedby trituration to release single cells.

We cultured the cell suspension, as previously described, innon-adherent vessels in the presence of EGF and FGF2. FIG. 23demonstrates that skin-derived stem cells harvested from human foreskinproliferate as non-adherent clusters. The clusters are morphologicallyindistinguishable from skin-derived stem cells derived from rodenttissue. Skin-derived clusters are loosely compacted, and the cells canbe readily dissociated manually without the use of proteases. Suchcharacteristics appear to distinguish skin-derived clusters fromCNS-derived neurospheres. Note that we have cultured these proliferatingskin-derived stem cells in the presence and absence of LIF, and haveobserved no significant differences in their proliferation,differentiation or survival characteristics. Furthermore, we havecurrently passaged and maintained foreskin derived stem cells asproliferating cultures for greater than three months.

One of the characteristics of skin-derived stem cells isolated fromrodents or other human tissue is the differentiation capacity of thesecells. We have previously demonstrated that skin-derived stem cells candifferentiate along a range of neuronal and non-neuronal fates.Similarly, foreskin-derived stem cells can differentiate along a widerange of neuronal and non-neuronal fates.

The expression of various neural and non-neural markers was assayedfollowing differentiation of foreskin-derived stem cells. Briefly,proliferating, non-adherent clusters were dissociated and plated on anadherent substratum in the presence of proliferation medium. Afterseveral days, the medium was changed to differentiation medium (5% fetalbovine serum/no mitogens), and marker expression was analyzed duringthis differentiation phase. Our results indicate that foreskin derivedstem cells can differentiate along a wide range of neuronal andnon-neuronal cell types. These results are consistent with our findingfor the differentiation potential of skin-derived stem cells obtainedfrom rodents and other human tissue samples. For example, underdifferentiation conditions, foreskin derived stem cells can expressnestin, fibronectin, bIII-tubulin, neurofilament-M, GFAP, CNP, S100,peripherin, and smooth muscle actin (FIGS. 23-27). The expression ofbIII-tubulin and neurofilament-M, in combination with the morphology ofthese cells, is indicative of the formation of highly complex neurons(FIG. 24). The expression of GFAP and CNP demonstrate the ability togive rise to glial cell types (FIG. 25). The expression of S100 andperipherin indicates that foreskin-derived stem cells can generateadditional neuronal cell types including bipolar cells (S100) andperipheral neurons (peripherin) (FIG. 26). Finally, the expression ofthe non-neural marker smooth muscle actin (FIG. 27) indicates that, ashas been observed for other skin-derived stem cells, foreskin-derivedstem cells have extensive differentiation capacity and can give rise toboth neural and non-neural cell types.

The foregoing experiments were performed using the following methods,except where otherwise noted.

Skin-Derived MSC Culture

For neonatal (three to 14 days) and adult (two months to one year) mice,skin from abdomen and back was carefully dissected free of other tissue,cut into 2-3 mm³ pieces, washed three times in HBSS, and then digestedwith 0.1% trypsin for 40 minutes at 37 C, followed by 0.1% DNAase forone minute at room temperature. Tissue pieces were then washed twicewith HBSS, once with media (DMEM-F12, 3:1, 1 g/ml fungizone, 1%penicillin/streptomycin) containing 10% rat serum (Harlan Bioproducts),and twice with serum-free media. Skin pieces were then mechanicallydissociated in media, and the suspension poured through a 40 M cellstrainer (Falcon). Dissociated cells were centrifuged, and resuspendedin 10 ml media containing B-27 supplement, 20 ng/ml EGF and 40 ng/mlbFGF (both Collaborative Research). Cells were cultured in 25 cm² tissueculture flasks (Corning) in a 37 C, 5% CO₂ tissue culture incubator.

To culture human skin-derived MSCs, two to three pieces of scalp tissueranging between 4-9 mm² (generated by placement of the stereotaxicapparatus for neurosurgery) were washed with HBSS, any subcutaneoustissue was removed, and the skin was cut into small pieces 1-2 mm³ insize. Tissue pieces were transferred to 15 mL Falcon tubes, washed threetimes with HBSS, and enzymatically digested in 0.1% trypsin for 40minutes at 37 C, and then washed as for mouse tissue. Dissociated cellswere suspended in 5 mL of the same media used for mouse cultures, withthe addition of 20 ng/ml LIF (R&D Systems Inc.). The cell suspension wasplaced in Falcon 6-well tissue culture plates and maintained in a 37 C,5% CO₂ tissue culture incubator. Cells were subcultured by partialdissociation of the clusters that formed every 7 to 10 days.

To passage floating clusters of cells, the medium containing the cellclusters was centrifuged, the cell pellet mechanically dissociated witha fire-polished Pasteur pipette, and the cells reseeded in fresh mediacontaining B-27 supplement and growth factors as above. Cells werepassaged every 6 to 7 days. For induction of differentiation into smoothmuscle cells, the cell clusters were centrifuged, the growthfactor-containing supernatant removed, and the clusters resuspended infresh media containing B-27 supplement and either 3% rat serum or 1-3%fetal bovine serum. The clusters were then plated onto 4-well Nunclonculture dishes coated with poly-D-lysine/laminin, and the medium waschanged every 3 to 7 days.

Transplantation of Olfactory Epithelium-Derived MSCs

Olfactory epithelium-derived MSCs were purified and cultured asdescribed herein. Female Sprague-Dawley rats or CD1 albino mice (CharlesRiver, Montreal, Quebec, Canada) weighing 180-200 g or 25-30 g,respectively, were anaesthetized with a mixture of ketamine (90 mg/kg)and xylazine (10 mg/kg) (intraperitoneal) prior to stereotacticinjections of 24 μg of 6-hydroxydopamine hydrobromide (dissolved in 5 μLof 0.9% saline containing 0.2 mg/ml ascorbate) into the right medialforebrain bundle (Tooth bar: −2.4 mm; A: −4.4 mm; L: 1.0 mm; V: 7.5 mm).Two weeks after the lesion, animals were tested for rotational behavior.Animals were immunosuppressed with cyclosporine (40 mg/kg,intraperitoneal) once a day until the day of sacrifice. For MSCtransplantation, anaesthetized animals were mounted in a Kopfstereotactic apparatus, and 2×2.5 μL aliquots of MSCs were injectedunilaterally into the lesioned caudate putamen or bilaterally in someanimals. The injections were made using a 5 μL Hamilton syringe at thefollowing coordinates: Tooth bar, −2.4 mm; A: 0.2; L: 3.0; V: 5.5-6.0.Injections were performed over a period of three minutes, a further fiveminutes was allowed for diffusion, and the needle was then retracted.These 5 μL injections contained MSCs derived from one neonatal pupcultured for 7 to 14 days. For the BrdU experiments, BrdU (10 μM) wasadded to culture media for 18 hours, after which the MSCs were washedthree times with fresh media to remove the BrdU, and then transplantedone day later.

Transplantation of Skin-Derived MSCs

Labeling of skin-derived MSCs was performed as follows. Three days priorto transplantation, free-floating cell clusters were partiallydissociated by gentle trituration, and then exposed to 50 MOI of arecombinant adenovirus expressing GFP, using standard techniques.Twenty-four hours later, the MSCs were centrifuged, washed, andresuspended in fresh medium containing 2 μM BrdU for an additional twodays. Prior to transplantation, MSCs were rinsed five times with freshmedium and resuspended to a concentration of 50,000 cells/μl. At thetime of transplantation, approximately 75% of the MSCs expressed GFP,while 95% were BrdU positive.

MSCs labeled with BrdU and GFP were stereotaxically injected into theright lateral ventricle of cryoanaesthetized two day old rat pups(co-ordinates from Bregma: lateral 1.5 mm, ventral 3.3 mm).Approximately 50,000 cells were injected over a three minute period in avolume of 1 μL. Fourteen days following transplantation, mice wereperfused with 50 mL 4% formaldehyde buffered with PBS. Fifty microncoronal sections through the forebrain were cut using a freezingmicrotome and analyzed immunocytochemically. All eight animals receivingcell transplants showed extensive labeling for tagged cells. No evidenceof tumor formation was observed.

Immunostaining

Immunostaining of olfactory epithelium-derived MSCs was performed asfollows. With the exception of GC immunocytochemistry, culture disheswere washed twice with Tris-buffered saline (TBS; 10 mM Tris, 150 mMNaCl, pH 8), then fixed with 4% formaldehyde, washed three times withTBS, blocked with TBS plus 2% goat serum (Jackson ImmunoResearch,Mississuagua, Ontario, Canada), and 0.1% Triton-X (Sigma Chemicals, St.Louis Mo.) for 30 minutes, then incubated with primary antibody in TBSplus 2% goat serum. Following primary antibody incubation, the disheswere washed three times with TBS, incubated in secondary antibody in TBSplus 2% goat serum, washed three times, and then viewed with afluorescence inverted microscope. The antibodies to GFAP (BoehringerMannheim, Laval, Quebec, Canada), βIII tubulin (Sigma), NeuN (Dr. R.Mullen), MAP-2 (clone AP-20; Sigma), and NF-160 (American Tissue CultureCollection, Manassas Va.) were monoclonal antibodies used atconcentrations of 1:200; 1:25; 1:10, and 1:1 respectively. Antibodies tonestin (a gift from Dr. Ron MacKay (National Institutes of Health), TH(Eugenetech Eugene, Oreg.), and DBH (Eugenetech) were rabbit polyclonalantibodies used at concentrations of 1:1000, 1:200, and 1:200respectively. Secondary antibodies Cy3 conjugated goat anti-mouse(Jackson ImmunoResearch) and Cy3 conjugated goat anti-rabbit (JacksonImmunoResearch), and were used at 1:1500. For double-labellingexperiments, we used FITC goat anti-mouse (Jackson ImmunoResearch).

For GC immunocytochemistry, living cultures were incubated in DMEMcontaining HEPES, 5% heat inactivated horse serum (HS), and 1:10 GCantibody for 30 min at 37 C, washed three times with themedium/HEPES/HS, fixed with 4% formaldehyde for 15 minutes, rinsed threetimes in TBS, incubated in Cy3 conjugated goat anti-mouse antibody(1:1500) for two hours, and finally washed three times in TBS. Culturesprocessed for immunocytochemistry without primary antibodies revealed nostaining.

Immunocytochemical analysis of cultured skin-derived MSCs was performedas follows. The primary antibodies that were used were: anti-nestinpolyclonal (1:250, Dr. Ron McKay, NINDS), anti-nestin monoclonal (1:400,PharMingen Inc.), anti-βIII-tubulin monoclonal (1:500, Tuj1 clone,BabCo), anti-neurofilament-M polyclonal (1:200, Chemcon Intl.), anti-GADpolyclonal (1:800, Chemicon Intl.), anti-NSE polyclonal (1:2000,Polysciences Inc.), anti-GFAP polyclonal (1:200, DAKO), anti-CNPasemonoclonal (1:400, Promega), anti-p75NTR polyclonal (1:500, Promega),anti-SMA monoclonal (1:400, Sigma-Aldrich), and anti-A2B5 monoclonal(Dr. Jack Snipes, M.N.I.). The secondary antibodies were Cy3-conjugatedgoat anti-mouse (1:200), Cy3-conjugated goat anti-rabbit (1:400),FITC-conjugated goat anti-mouse (1:50-1:100), and FITC-conjugated goatanti-rabbit (1:200) (all from Jackson Immunoresearch Laboratories).

Immunocytochemical analysis of free-floating brain sections wasperformed by DAB immunohistochemistry. For GFP, sections were incubatedin 0.3% H₂O₂ for one hour to inhibit endogenous tissue peroxidaseactivity prior to blocking. For BrdU immunohistochemistry, sections werepre-incubated in 0.5% sodium borohydride for 20 minutes prior toblocking of endogenous peroxidase activity in 0.03% H₂O₂ for 30 minutes.To permeabilize the nuclei for BrdU immunohistochemistry, sections wereincubated in 1% DMSO for 10 minutes, the DNA denatured with 2N HCl for60 minutes, and the HCl neutralized with 0.1M borate buffer for 5minutes. All sections were blocked for one hour in 10% BSA, and thenincubated for 48 hours at 4° C. with either anti-GFP (1:1000, Clontech)or anti-BrdU (1:100, Becton-Dickinson). Primary antibodies were detectedusing a biotinylated horse anti-mouse secondary antibody (1:200, VectorLaboratories) for one hour at room temperature, and visualized using theVectastain kit (Vector Laboratories) and a nickel-enhanced DAB reactioncontaining 0.05% DAB, 0.04% nickel chloride, and 0.015% H₂O₂. Sectionswere mounted onto slides, dehydrated through a series of ethanols andHistoclear (Fisher Scientific), and coverslipped using Permount (FisherScientific).

Fluorescence immunohistochemistry was performed to co-localize GFPexpression with NSE. Free-floating sections were blocked in 10% BSA forone hour at room temperature, and then incubated 48 hours at 4° C. in asolution containing mouse anti-GFP and rabbit anti-NSE. Sections wereincubated with Cy3 conjugated anti-mouse and FITC conjugated anti-rabbitsecondary antibodies for one hour at room temperature, and coverslippedusing Sigma Mounting Medium.

Other Embodiments

The present invention has been described in terms of particularembodiments found or proposed by the present inventors to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. All such modifications are intended to beincluded within the scope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

1. A cellular composition comprising a purified population ofmultipotent mammalian cells from skin, which multipotent cells formnon-adherent clusters in culture, are self renewing, are positive fornestin and fibronectin protein, and differentiate into both neuronal andnon-neuronal cell types.
 2. The cellular composition of claim 1, whereinsaid cells are capable of differentiating into a non-neural cell type.3. The cellular composition of claim 1, wherein said cells canproliferate in culture in the absence of exogenous LIF.
 4. The cellularcomposition of claim 1, wherein said cells are negative for vimentin andcytokeratin as measured by immunohistochemistry.
 5. A cellularcomposition comprising multipotent mammalian cells from skin with fewerthan 30 percent lineage committed cells, wherein said multipotent cellsform non-adherent clusters in culture, are self renewing, are positivefor nestin and fibronectin protein, and differentiate into ectodermaland mesodermal cell types.
 6. A cellular composition comprising apurified population of multipotent cells from skin prepared by themethod comprising: (a) culturing a dissociated sample of epithelialtissue; (b) isolating, from the culture, non-adherent cellscharacterized by the following: are positive for nestin and fibronectinprotein, are self renewing, and differentiate into ectodermal andmesodermal cell types.
 7. A cellular composition comprising a purifiedpopulation of multipotent cells from skin prepared by the methodcomprising: (a) separating the dermal and epidermal layers of epithelialtissue; (b) culturing a dissociated sample of the dermal layer of saidepithelial tissue; (c) isolating, from the culture, non-adherent cellscharacterized by the following: are self-renewing, and differentiateinto ectodermal and mesodermal cell types.
 8. A cellular compositioncomprising a purified population of multipotent mammalian cells fromskin, which multipotent cells form non-adherent clusters in culture, areself-renewing, are positive for nestin and fibronectin protein,differentiate into both neuronal and non-neuronal cell types, and canproliferate in culture in the absence of exogenous EGF.
 9. A cellularcomposition comprising a purified population of multipotent mammaliancells from skin, which multipotent cells form non-adherent clusters inculture, are self-renewing, are positive for nestin and fibronectinprotein, are negative for vimentin and cytokeratin protein, anddifferentiate into both neuronal and non-neuronal cell types.
 10. Acellular composition comprising a purified population of multipotentmammalian cells from skin, which multipotent cells form non-adherentclusters in culture, are self-renewing, are positive for nestin andfibronectin protein, are negative for vimentin, cytokeratin, and p75protein, and differentiate into both neuronal and non-neuronal celltypes.
 11. The cellular composition of any of claims 8-9, and 10 whichmultipotent cells can differentiate into cells expressing one or moremarkers selected from the group consisting of Glial Fibrillary AcidProtein (GFAP), neurofilament 160, βIII tubulin, NeuN, neurofilament-M(NFM), neuron-specific enolase, galactocerebroside, GAD, tyrosinehydroxylase (TH), dopamine β-dehydrogenase, and CNPase.
 12. The cellularcomposition of any of claims 8-9, and 10, which multipotent cells candifferentiate to form cells selected from the group consisting ofepithelial cells, endothelial cells, skeletal muscle cells, cardiacmuscle cells, connective tissue cells, lung cells, adipocytes,pancreatic islet cells, hematopoietic cells, chondrocytes, bone, kidneycells, and hepatocytes.